技术领域Technical Field
本发明涉及生物技术领域，具体涉及一种成体干细胞来源的细胞内纳米囊泡在治疗眼科疾病中的应用，特别是间充质干细胞来源的细胞内纳米囊泡在治疗角膜损伤、视网膜损伤和变性中的应用。The present invention relates to the field of biotechnology, and in particular to an application of intracellular nanovesicles derived from adult stem cells in treating ophthalmic diseases, in particular to an application of intracellular nanovesicles derived from mesenchymal stem cells in treating corneal damage, retinal damage and degeneration.
背景技术Background Art
角膜是眼球壁外层前部的透明部分，在透光和保护眼内容物方面起着重要作用。角膜暴露在外部环境下，因此容易受到损伤和感染。由于角膜有密集的神经支配，持续的角膜损伤可能会引起疼痛，导致不可逆的角膜混浊，致使视力模糊、减退，甚至失明。角膜损伤愈合是一个复杂的、多组织协调的过程，涉及上皮层的修复、存活的上皮细胞和成纤维细胞迁移使伤口闭合，以及刺激细胞增殖使组织再生。为了避免纤维化和血管生成损害角膜的透明度，防止过度的基质肌成纤维细胞活化和血管新生也是必要的。复杂角膜损伤的当前标准治疗包括最大限度地使用局部润滑剂、最大限度地减少泪液蒸发、局部应用抗生素、用绷带式隐形眼镜保护角膜表面以及手术治疗。然而，即使联合应用这些措施治疗效果也是甚微的。此外，已知的眼部用药常见副作用通常包括过敏反应、刺激感、瘙痒、水肿、目赤、损伤延迟愈合、眼压增加、青光眼恶化、白内障形成和视神经损伤。The cornea is the transparent part of the outer layer of the eyeball wall, which plays an important role in transmitting light and protecting the contents of the eye. The cornea is exposed to the external environment and is therefore susceptible to injury and infection. Because the cornea is densely innervated, persistent corneal damage may cause pain and lead to irreversible corneal opacity, resulting in blurred vision, decreased vision, and even blindness. Corneal injury healing is a complex, multi-tissue coordinated process involving the repair of the epithelial layer, the migration of surviving epithelial cells and fibroblasts to close the wound, and the stimulation of cell proliferation to regenerate the tissue. In order to avoid fibrosis and angiogenesis that impair corneal transparency, it is also necessary to prevent excessive stromal myofibroblast activation and angiogenesis. The current standard treatment of complex corneal injuries includes maximizing the use of topical lubricants, minimizing tear evaporation, topical antibiotics, protecting the corneal surface with bandage contact lenses, and surgical treatment. However, even the combined use of these measures has little therapeutic effect. In addition, known common side effects of ocular medications generally include allergic reactions, irritation, itching, edema, eye redness, delayed healing of injuries, increased intraocular pressure, worsening of glaucoma, cataract formation, and optic nerve damage.
视网膜是眼球的重要组成部分，承担光电信号转换功能，其损伤和变性会导致不可逆的视力下降。例如，视网膜色素变性，黄斑变性和遗传性视网膜营养不良等。这些疾病的特点是视网膜细胞的凋亡及功能丧失。其中光感受器细胞包括视锥和视杆细胞，对于维持视网膜光电信号转化十分重要，但是强光照射或者遗传因素会导致光感受器细胞凋亡，视网膜外核层逐渐消失，进而导致视网膜功能障碍。此外，视网膜色素上皮细胞（pigmentepithelial cells, RPE）具有复杂的生物学功能，可为神经视网膜的外层细胞提供营养、吞噬和消化光感受器细胞外节盘膜，从而维持神经视网膜正常新陈代谢。RPE细胞的损伤可引起RPE-玻璃膜-脉络膜毛细血管复合体的损害，导致眼底疾病的发生，如年龄相关性黄斑变性，视网膜色素变性和各种脉络膜视网膜病变等。蓝光照射产生视网膜损伤的动物模型模拟了视网膜光感受器细胞凋亡的病理改变，可导致一系列视网膜功能障碍，包括电生理反应中a波和b波潜伏期延长，振幅下降；RPE内线粒体肿胀，出现空泡；视网膜光感受器外节膜盘大量脱落，空泡变性等，是常见的模拟视网膜损伤及变性类疾病的模型。Rd10是常用的模拟视网膜色素变性的小鼠模型，其特点为遗传缺陷导致的视网膜色素变性。其自出生后第14天起，视网膜的外核层逐渐变薄，外核层与内核层之间的突触连接逐渐消失，电生理反应逐渐减少，整个病理过程伴随着细胞氧化应激水平升高以及大量的视网膜细胞凋亡。Rd10是常见的遗传性视网膜病变的模型，可以模拟以视网膜氧化应激和炎症等病理改变为特征的疾病。The retina is an important part of the eyeball and is responsible for the conversion of photoelectric signals. Its damage and degeneration can lead to irreversible vision loss. For example, retinitis pigmentosa, macular degeneration and hereditary retinal dystrophy. These diseases are characterized by apoptosis and functional loss of retinal cells. Among them, photoreceptor cells include cones and rods, which are very important for maintaining the conversion of retinal photoelectric signals. However, strong light exposure or genetic factors can cause photoreceptor cell apoptosis, the retinal outer nuclear layer gradually disappears, and then lead to retinal dysfunction. In addition, retinal pigment epithelial cells (RPE) have complex biological functions, which can provide nutrition for the outer cells of the neural retina, phagocytose and digest the outer segment disc membrane of photoreceptor cells, thereby maintaining the normal metabolism of the neural retina. Damage to RPE cells can cause damage to the RPE-vitreous membrane-choroidal capillary complex, leading to the occurrence of fundus diseases, such as age-related macular degeneration, retinitis pigmentosa and various chorioretinopathy. The animal model of retinal damage caused by blue light irradiation simulates the pathological changes of retinal photoreceptor cell apoptosis, which can lead to a series of retinal dysfunctions, including prolonged latency and decreased amplitude of a-wave and b-wave in electrophysiological response; mitochondrial swelling and vacuoles in RPE; massive shedding of retinal photoreceptor outer segment membrane discs and vacuolar degeneration, etc. It is a common model for simulating retinal damage and degenerative diseases. Rd10 is a commonly used mouse model for simulating retinitis pigmentosa, which is characterized by retinitis pigmentosa caused by genetic defects. Since the 14th day after birth, the outer nuclear layer of the retina gradually becomes thinner, the synaptic connection between the outer nuclear layer and the inner nuclear layer gradually disappears, the electrophysiological response gradually decreases, and the entire pathological process is accompanied by increased cellular oxidative stress levels and a large number of retinal cell apoptosis. Rd10 is a common model of hereditary retinopathy, which can simulate diseases characterized by pathological changes such as retinal oxidative stress and inflammation.
干细胞如间充质干细胞（mesenchymal stem cells，MSCs）的细胞疗法已成为眼科疾病的一种有前途的治疗手段。这种治疗方法取决于间充质干细胞转分化为神经细胞的能力，以及它们的自我更新潜力、促增殖特性。然而，细胞治疗在眼科疾病的应用存在较多瓶颈，包括细胞异常生长和免疫排斥等。尽管干细胞分泌的细胞外囊泡（ExtracellularVesicles, EVs）具有干细胞特性，可以代替细胞发挥治疗作用，但其仍然存在一定的缺陷，例如细胞外囊泡的收集效率低，细胞外囊泡被分泌后存在于细胞外基质中，其含有细胞培养基中的外源性物质导致纯度下降。Cell therapy using stem cells, such as mesenchymal stem cells (MSCs), has become a promising treatment for ophthalmic diseases. This treatment method depends on the ability of mesenchymal stem cells to transdifferentiate into neural cells, as well as their self-renewal potential and pro-proliferation properties. However, there are many bottlenecks in the application of cell therapy in ophthalmic diseases, including abnormal cell growth and immune rejection. Although extracellular vesicles (EVs) secreted by stem cells have stem cell properties and can replace cells to play a therapeutic role, they still have certain defects, such as low efficiency of extracellular vesicle collection, extracellular vesicles are present in the extracellular matrix after being secreted, and they contain exogenous substances in the cell culture medium, resulting in reduced purity.
值得注意的是，在细胞内还有许多纳米级别的囊泡，它们位于富含膜的多种细胞器之间，负责细胞内物质运输和分泌途径。细胞内囊泡（Intracellular Nanovesicles,IVs）通过一种被称为小泡出芽的过程产生，可以起源于各种细胞器，包括内质网、高尔基体、内体和质膜。这些IVs维持了细胞内的基础生命活动，其内含有大量的生物大分子，其通过胞吐作用参与特定蛋白质、激素和其他生物分子的分泌。一种分泌囊泡亚型，也被称为突触囊泡。现有技术中尚无关于细胞内纳米囊泡在眼科疾病中应用的报道。It is worth noting that there are many nano-scale vesicles in the cell, which are located between various membrane-rich organelles and are responsible for the intracellular material transport and secretion pathways. Intracellular vesicles (IVs) are produced by a process called vesicle budding and can originate from various organelles, including the endoplasmic reticulum, Golgi apparatus, endosomes, and plasma membranes. These IVs maintain the basic life activities in the cell, contain a large number of biological macromolecules, and participate in the secretion of specific proteins, hormones, and other biological molecules through exocytosis. A subtype of secretory vesicles, also known as synaptic vesicles. There are no reports in the prior art on the application of intracellular nanovesicles in ophthalmic diseases.
发明内容Summary of the invention
为克服现有技术的不足，本发明提供了一种成体干细胞（特别是间充质干细胞）来源的细胞内纳米囊泡在治疗眼科疾病（特别是，角膜损伤、视网膜损伤和变性）中的应用。To overcome the deficiencies of the prior art, the present invention provides an application of intracellular nanovesicles derived from adult stem cells (particularly mesenchymal stem cells) in the treatment of ophthalmic diseases (particularly corneal damage, retinal damage and degeneration).
本发明的第一方面，提供一种成体干细胞来源的细胞内纳米囊泡在制备预防和/或治疗眼科疾病的药物中的应用。The first aspect of the present invention provides a use of intracellular nanovesicles derived from adult stem cells in the preparation of a drug for preventing and/or treating ophthalmic diseases.
具体地，所述囊泡为小细胞内纳米囊泡（small Intracellular Vesicles,sIVs）。Specifically, the vesicles are small intracellular nanovesicles (sIVs).
具体地，所述囊泡呈双层马蹄形或茶杯型。Specifically, the vesicle is double-layered horseshoe-shaped or teacup-shaped.
具体地，所述囊泡的平均粒径为50-100nm（例如50、55、60、65、70、75、80、85、90、95、100nm），特别是65-85nm。Specifically, the average particle size of the vesicles is 50-100 nm (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 nm), in particular 65-85 nm.
具体地，所述囊泡表达TMEM214蛋白。Specifically, the vesicles express TMEM214 protein.
具体地，所述囊泡低表达外泌体标志物，高表达细胞内富膜细胞器的标志蛋白和Clathrin蛋白家族。Specifically, the vesicles lowly express exosome markers, and highly express marker proteins of intracellular membrane-rich organelles and the Clathrin protein family.
具体地，所述囊泡在-80℃至37℃（例如-80、-50、-20、-10、0、5、25、37℃）下均较稳定。Specifically, the vesicles are relatively stable at -80°C to 37°C (e.g., -80, -50, -20, -10, 0, 5, 25, 37°C).
具体地，所述囊泡通过包含超声处理步骤的方法制得。更具体地，所述囊泡通过包含超声处理、离心处理、超速离心处理步骤（依次进行）的方法制得。Specifically, the vesicles are prepared by a method comprising an ultrasonic treatment step. More specifically, the vesicles are prepared by a method comprising an ultrasonic treatment, a centrifugation treatment, and an ultracentrifugation treatment step (performed sequentially).
在本发明的一个优选实施方式中，所述囊泡通过包含以下步骤的方法制得：In a preferred embodiment of the present invention, the vesicles are prepared by a method comprising the following steps:
（1）取成体干细胞分散在悬浮溶剂中，进行超声处理；(1) Adult stem cells are dispersed in a suspension solvent and subjected to ultrasonic treatment;
（2）将步骤（1）所得液体进行一次或多次离心处理，弃去细胞膜及细胞器碎片，取上清液；(2) centrifuging the liquid obtained in step (1) once or multiple times, discarding cell membranes and organelle fragments, and taking the supernatant;
（3）将步骤（2）所得上清液进行超速离心处理，取沉淀为细胞内纳米囊泡；(3) subjecting the supernatant obtained in step (2) to ultracentrifugation to obtain the precipitate as intracellular nanovesicles;
任选地，（4）将步骤（3）所得沉淀重悬。Optionally, (4) resuspending the precipitate obtained in step (3).
具体地，步骤（1）中所述细胞为经过培养、消化、洗涤后所得分离的细胞（其经过弃去细胞培养基、消化、洗涤等步骤，可排除分离获得细胞外囊泡的可能性）。Specifically, the cells in step (1) are cells isolated after culture, digestion and washing (after discarding the cell culture medium, digestion, washing and other steps, the possibility of obtaining extracellular vesicles by separation can be eliminated).
具体地，所述方法还可以包括细胞消化、计数步骤；在本发明的一些实施方案中，所述细胞消化的步骤包括：培养细胞生长至90%融合，弃去细胞培养基，洗涤细胞，加入胰酶消化细胞，然后中和并清洗细胞。Specifically, the method may further include cell digestion and counting steps; in some embodiments of the present invention, the cell digestion step includes: culturing cells to grow to 90% confluence, discarding the cell culture medium, washing the cells, adding trypsin to digest the cells, and then neutralizing and washing the cells.
具体地，所述细胞在悬浮溶剂中的细胞密度为1-4×106个/mL，例如1×106个/mL、2×106个/mL、3×106个/mL、4×106个/mL。在本发明的一些实施方案中，所述细胞密度为1×106个/mL。Specifically, the cell density of the cells in the suspension solvent is 1-4×106 cells/mL, such as 1×106 cells/mL, 2×106 cells/mL, 3×106 cells/mL, 4×106 cells/mL. In some embodiments of the present invention, the cell density is 1×106 cells/mL.
具体地，所述悬浮溶剂为任何适于培养细胞的缓冲液，例如PBS、Tris缓冲液、甘氨酸缓冲液。在本发明的一些实施方案中，所述溶剂为PBS。Specifically, the suspension solvent is any buffer suitable for culturing cells, such as PBS, Tris buffer, glycine buffer. In some embodiments of the present invention, the solvent is PBS.
具体地，所述成体干细胞是指存在于一种已经分化组织中的未分化细胞，这种细胞能够自我更新并且能够特化形成组成该类型组织的细胞，在特定条件下，成体干细胞或者产生新的干细胞，或者按一定的程序分化，形成新的功能细胞，从而使组织和器官保持生长和衰退的动态平衡。例如，间充质干细胞（MSC）、造血干细胞（HSC）、神经干细胞（NSC）等，特别是间充质干细胞。Specifically, the adult stem cells refer to undifferentiated cells existing in a differentiated tissue, which can self-renew and specialize to form cells that make up this type of tissue. Under certain conditions, adult stem cells either produce new stem cells or differentiate according to a certain procedure to form new functional cells, thereby maintaining a dynamic balance of growth and decline of tissues and organs. For example, mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells (NSC), etc., especially mesenchymal stem cells.
具体地，所述成体干细胞（特别是间充质干细胞）来源于哺乳动物，特别是人类。Specifically, the adult stem cells (especially mesenchymal stem cells) are derived from mammals, especially humans.
具体地，所述间充质干细胞选自：脐带间充质干细胞（UC-MSC）、骨髓间充质干细胞（BM-MSC）、脂肪间充质干细胞（AD-MSC）、牙髓间充质干细胞、胎盘和羊水以及羊膜间充质干细胞，特别是脐带间充质干细胞（UC-MSC）。Specifically, the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).
具体地，步骤（1）中所述超声处理的振幅为20%-25%（例如20%、22%、24%、25%）；在本发明的一些实施方案中，所述超声处理的振幅为20%。Specifically, the amplitude of the ultrasonic treatment in step (1) is 20%-25% (e.g., 20%, 22%, 24%, 25%); in some embodiments of the present invention, the amplitude of the ultrasonic treatment is 20%.
具体地，步骤（1）中所述超声处理的时间为10-20s（例如10、15、18、20s），优选为15s；在本发明的一些实施方案中，所述超声处理的时间为15s，on 2s，off 2s。Specifically, the ultrasonic treatment time in step (1) is 10-20 s (e.g., 10, 15, 18, 20 s), preferably 15 s. In some embodiments of the present invention, the ultrasonic treatment time is 15 s, on 2 s, off 2 s.
在本发明的一些实施方案中，步骤（2）中所述离心处理的次数为两次，其各自参数分别为：In some embodiments of the present invention, the number of centrifugation treatments in step (2) is two, and the respective parameters are:
1000-3000g（例如1000、1500、2000、2500、3000g），5-20分钟（例如5、8、10、12、15、20分钟）；1000-3000 g (e.g. 1000, 1500, 2000, 2500, 3000 g), 5-20 minutes (e.g. 5, 8, 10, 12, 15, 20 minutes);
10000-30000g（例如10000、15000、20000、25000、30000g），20-40分钟（例如20、25、28、30、32、35、40分钟）。10000-30000 g (e.g. 10000, 15000, 20000, 25000, 30000 g), 20-40 min (e.g. 20, 25, 28, 30, 32, 35, 40 min).
在本发明的一个实施方案中，第一次离心在2000g下进行10分钟。In one embodiment of the invention, the first centrifugation is performed at 2000 g for 10 minutes.
在本发明的一个实施方案中，第二次离心在20000g下进行30分钟。In one embodiment of the invention, the second centrifugation is performed at 20000 g for 30 minutes.
具体地，步骤（3）中所述超速离心处理的参数包括100000-180000g（例如100000、120000、140000、150000、160000、180000g），50-100分钟（例如50、60、65、70、75、80、90、100分钟）。Specifically, the parameters of the ultracentrifugation treatment in step (3) include 100,000-180,000 g (e.g., 100,000, 120,000, 140,000, 150,000, 160,000, 180,000 g), 50-100 minutes (e.g., 50, 60, 65, 70, 75, 80, 90, 100 minutes).
在本发明的一个实施方案中，所述超速离心在150000g下进行70分钟。In one embodiment of the invention, the ultracentrifugation is performed at 150000 g for 70 minutes.
具体地，步骤（4）中所述重悬溶剂为任何适于培养细胞的缓冲液，例如PBS、Tris缓冲液、甘氨酸缓冲液。在本发明的一些实施方案中，所述重悬溶剂为PBS。Specifically, the resuspension solvent in step (4) is any buffer suitable for culturing cells, such as PBS, Tris buffer, glycine buffer. In some embodiments of the present invention, the resuspension solvent is PBS.
具体地，所述超声处理、离心处理、超速离心处理中的一种或多种在低温下操作，例如0-5℃（例如0、1、2、3、4、5℃）下；特别是，所述超声处理、离心处理、超速离心处理均在4℃或在冰上进行。Specifically, one or more of the ultrasonic treatment, centrifugation treatment and ultracentrifugation treatment are performed at low temperature, such as 0-5°C (e.g. 0, 1, 2, 3, 4, 5°C); in particular, the ultrasonic treatment, centrifugation treatment and ultracentrifugation treatment are all performed at 4°C or on ice.
在本发明的一些实施方案中，所述囊泡通过包含以下步骤的方法制得：取1×106个/mL密度的细胞悬液，将超声探头放入液面中央，超声振幅参数范围是20%，时间参数范围是15s，on 2s，off 2s，进行超声处理；然后将该液体转移至离心管进行离心，离心参数是2000g×10min，20000g×30min，收集上清液；将该上清液转移至超速离心管进行离心，离心参数是150000g×70min。In some embodiments of the present invention, the vesicles are prepared by a method comprising the following steps: taking a cell suspension with a density of 1×106 cells/mL, placing an ultrasonic probe in the center of the liquid surface, and performing ultrasonic treatment with an ultrasonic amplitude parameter range of 20% and a time parameter range of 15s, on 2s, and off 2s; then transferring the liquid to a centrifuge tube for centrifugation with centrifugation parameters of 2000g×10min and 20000g×30min, and collecting the supernatant; transferring the supernatant to an ultracentrifuge tube for centrifugation with centrifugation parameters of 150000g×70min.
在本发明的一些实施方案中，所述眼科疾病为角膜疾病。In some embodiments of the invention, the ophthalmic disease is a corneal disease.
具体地，所述角膜疾病选自：角膜炎（如感染性角膜炎（如疱疹性角膜炎）、外伤性角膜炎、自身免疫性角膜炎）、角膜溃疡（如中心性角膜溃疡、蚕蚀性角膜溃疡、边缘性角膜溃疡、前房积脓性角膜溃疡、细菌性角膜溃疡、病毒性角膜溃疡、环形角膜溃疡、角膜糜烂、角膜溃疡性穿孔）、角膜穿孔、浅层角膜炎（如晕性角膜炎、星状角膜炎、条纹状角膜炎、钱币状角膜炎、光敏性角膜炎、雪盲）、电光性眼炎、浅层点状角膜炎、丝状角膜炎、角膜结膜炎（如暴露性角膜结膜炎、结节性眼炎）、小泡性角膜结膜炎、神经营养性角膜结膜炎、浅层角膜结膜炎、暴露性角膜炎、基质层和深层角膜炎（如硬化性角膜炎、角膜基质炎）、深层角膜炎、角膜脓肿、科根综合征、角膜新生血管、角膜血管翳、角膜血管影、药物性角膜结膜炎、大泡性角膜炎、细菌性角膜炎、化脓性角膜炎、神经麻痹性角膜炎、角膜瘢痕和混浊、角膜瘢痕、角膜混浊、粘连性白斑、角膜瘢痕、角膜云翳、角膜斑翳、角膜色素沉着和沉着物（如角膜Kayser-Fleischer环、克鲁肯贝格梭、施特里线）、角膜沉着物、角膜黑变病、角膜血染、大泡性角膜病变、角膜水肿、角膜层改变、角膜变性（如角膜角化病、角膜软化症）、角膜老年环、带状角膜病变、Salzmann结节状角膜变性、边缘性角膜变性、滴状角膜、遗传性角膜营养不良（如颗粒状角膜基质营养不良、斑状角膜营养不良）、上皮基底膜营养不良、角膜营养不良（如Grayson-Wilbrandt角膜营养不良、Meesmann角膜营养不良、Schnyder角膜营养不良、Thiel-behnke角膜营养不良、X连锁角膜营养不良、后部无定形角膜营养不良、后弹力层角膜营养不良、胶滴状角膜营养不良）、格子状角膜营养不良、Fuchs角膜内皮营养不良、圆锥角膜、角膜畸形（如角膜突出）、角膜葡萄肿、角膜后弹性层膨出、角膜切口瘘、角膜炎性肿物、角膜上皮损伤、角膜知觉减退、角膜内皮炎、角膜溶解、角膜囊肿、角膜干燥症、复发性角膜糜烂、角膜皮赘、角膜上皮脱落、角膜结膜化、角膜内皮失代偿、角膜肿物、腺病毒性角膜结膜炎、流行性角膜结膜炎、角膜肿瘤（如角膜内上皮癌、角膜鳞状细胞癌、角膜原位癌）、白内障术后角膜病变、先天性角膜混浊、先天性角膜白斑、先天性角膜畸形（如先天性扁平角膜、先天性角膜异常、先天性角膜巩膜化）、先天性青光眼（如先天性球形角膜伴青光眼）、先天性眼畸形（如先天性大角膜、先天性小角膜、先天性球形角膜）、彼得异常、结合膜损伤和角膜擦伤、角膜擦伤、角膜磨损、角膜异物、角膜和结合膜囊烧伤、角膜烧伤、角膜和结合膜囊腐蚀伤、角膜腐蚀伤、角膜伴结膜酸性烧伤、角膜化学性烧伤、角膜碱性烧伤、角膜酸性烧伤、眼结核（如角膜结核）、晚期先天性梅毒性眼病（如晚期先天性梅毒性角膜炎、晚期先天性梅毒性间质性角膜炎）、沙眼（如沙眼性角膜炎）、疱疹病毒性眼病（如疱疹病毒性角膜炎、疱疹性角膜结膜炎）、带状疱疹眼病（如带状疱疹性角膜炎、带状疱疹性角膜结膜炎）、眼原位癌（如角膜原位癌）、眼部类天疱疮、糖尿病性角膜病变、虹膜角膜内皮综合征、角膜移植后的移植缺陷、Sjogren综合症、Stevens-Johnson综合症、青光眼、干性角结膜炎（如干眼病）、红眼病。Specifically, the corneal disease is selected from: keratitis (such as infectious keratitis (such as herpetic keratitis), traumatic keratitis, autoimmune keratitis), corneal ulcer (such as central corneal ulcer, Mooren's ulcer, marginal corneal ulcer, anterior chamber suppurative corneal ulcer, bacterial corneal ulcer, viral corneal ulcer, annular corneal ulcer, corneal erosion, corneal ulcer perforation), corneal perforation, superficial keratitis (such as halo keratitis, stellate keratitis, stripe keratitis, nummular keratitis , photosensitive keratitis, snow blindness), electric eye inflammation, superficial punctate keratitis, filamentary keratitis, keratoconjunctivitis (such as exposure keratoconjunctivitis, nodular eye inflammation), alveolar keratoconjunctivitis, neurotrophic keratoconjunctivitis, superficial keratoconjunctivitis, exposure keratitis, stromal and deep keratitis (such as sclerosing keratitis, stromal keratitis), deep keratitis, corneal abscess, Cogan's syndrome, corneal neovascularization, corneal pannus, corneal vascular shadow, drug-induced keratoconjunctivitis, bullous keratitis , bacterial keratitis, suppurative keratitis, neuroparalytic keratitis, corneal scars and opacities, corneal scars, corneal opacities, adhesive leukoplakia, corneal scars, corneal clouds, corneal leukoplakia, corneal pigmentation and deposits (such as corneal Kayser-Fleischer rings, Krukenberg shuttles, Stry lines), corneal deposits, corneal melanosis, corneal blood staining, bullous keratopathy, corneal edema, corneal layer changes, corneal degeneration (such as corneal keratosis, keratomalacia), cornea Senile ring, band keratopathy, Salzmann nodular corneal degeneration, marginal corneal degeneration, guttate keratopathy, hereditary corneal dystrophies (such as granular corneal stromal dystrophy, macular corneal dystrophy), epithelial basement membrane dystrophy, corneal dystrophies (such as Grayson-Wilbrandt corneal dystrophy, Meesmann corneal dystrophy, Schnyder corneal dystrophy, Thiel-behnke corneal dystrophy, X-linked corneal dystrophy, etc.) Locking corneal dystrophy, posterior amorphous corneal dystrophy, Descemet's corneal dystrophy, gelatinous corneal dystrophy), lattice corneal dystrophy, Fuchs corneal endothelial dystrophy, keratoconus, corneal deformity (such as corneal protrusion), corneal staphyloma, Descemet's membrane ectasia, corneal incision fistula, corneal inflammatory tumor, corneal epithelial damage, corneal hypoesthesia, corneal endotheliitis, corneal dissolution, corneal cyst, corneal sicca, recurrent corneal erosion, corneal skin tag, corneal epithelial detachment corneal conjunctivalization, corneal endothelial decompensation, corneal tumors, adenoviral keratoconjunctivitis, epidemic keratoconjunctivitis, corneal tumors (such as corneal epithelial carcinoma, corneal squamous cell carcinoma, corneal carcinoma in situ), corneal lesions after cataract surgery, congenital corneal opacity, congenital corneal leukoma, congenital corneal malformations (such as congenital flat cornea, congenital corneal abnormality, congenital corneal scleralization), congenital glaucoma (such as congenital spherical cornea with glaucoma), congenital eye malformations (such as congenital macrocornea, congenital Congenital microcornea, congenital spherical cornea), Peter anomaly, conjunctival injury and corneal abrasion, corneal abrasion, corneal abrasion, corneal foreign body, corneal and conjunctival sac burns, corneal burns, corneal and conjunctival sac erosions, corneal erosions, corneal and conjunctival acid burns, corneal chemical burns, corneal alkaline burns, corneal acid burns, ocular tuberculosis (such as corneal tuberculosis), late congenital syphilitic eye diseases (such as late congenital syphilitic keratitis, late congenital syphilitic interstitial keratitis), trachoma (such as Trachoma keratitis), herpes virus eye disease (such as herpes virus keratitis, herpetic keratoconjunctivitis), herpes zoster eye disease (such as herpes zoster keratitis, herpes zoster keratoconjunctivitis), carcinoma in situ of the eye (such as corneal carcinoma in situ), ocular pemphigoid, diabetic keratopathy, iridocorneal endothelial syndrome, graft defect after corneal transplantation, Sjogren's syndrome, Stevens-Johnson syndrome, glaucoma, keratoconjunctivitis sicca (such as dry eye), and conjunctivitis.
在本发明的一些优选实施例中，所述角膜疾病为角膜损伤、角膜新生血管、角膜炎、角膜营养不良。In some preferred embodiments of the present invention, the corneal disease is corneal injury, corneal neovascularization, keratitis, and corneal dystrophy.
具体地，所述角膜损伤可以是各种原因（如外力、化学刺激、感染、角膜接触镜佩戴不当、营养不良、其他角膜疾病如角膜肿瘤等）引起的角膜结构破坏，例如机械性角膜损伤、化学性角膜损伤（如角膜碱烧伤）等，其表现可包括，但不限于，角膜上皮损伤、角膜基质层水肿、角膜破裂、角膜知觉减退、角膜变薄、角膜脱离、角膜劈裂、角膜震荡、角膜的出血、渗血或者是缺血等。在本发明的一个实施例中，所述角膜损伤为机械性角膜损伤。在本发明另一个实施例中，所述角膜损伤为角膜碱烧伤。Specifically, the corneal injury may be corneal structural damage caused by various reasons (such as external force, chemical stimulation, infection, improper wearing of corneal contact lenses, malnutrition, other corneal diseases such as corneal tumors, etc.), such as mechanical corneal injury, chemical corneal injury (such as corneal alkali burns), etc., and its manifestations may include, but are not limited to, corneal epithelial injury, corneal stroma edema, corneal rupture, corneal hypoesthesia, corneal thinning, corneal detachment, corneal splitting, corneal concussion, corneal bleeding, oozing or ischemia, etc. In one embodiment of the present invention, the corneal injury is mechanical corneal injury. In another embodiment of the present invention, the corneal injury is corneal alkali burns.
在本发明的一些实施方案中，所述眼科疾病为视网膜疾病。In some embodiments of the invention, the ophthalmic disease is a retinal disease.
具体地，所述视网膜疾病为视网膜损伤和变性及其相关疾病。Specifically, the retinal disease is retinal damage and degeneration and related diseases.
具体地，所述视网膜损伤可以是各种原因引起的视网膜损伤，例如光化学损伤，其表现可包括，但不限于，视网膜变薄、视网膜脱离、视网膜劈裂、视网膜震荡、视网膜的出血、渗出或者是缺血等。Specifically, the retinal damage may be retinal damage caused by various reasons, such as photochemical damage, and its manifestations may include, but are not limited to, retinal thinning, retinal detachment, retinal splitting, retinal concussion, retinal hemorrhage, exudation or ischemia, etc.
具体地，所述视网膜损伤相关疾病包括，但不限于，光损伤性视网膜病变、青光眼、缺血性视网膜病变、糖尿病视网膜病变、等。Specifically, the retinal damage-related diseases include, but are not limited to, photoretinopathy, glaucoma, ischemic retinopathy, diabetic retinopathy, and the like.
具体地，所述视网膜变性相关疾病包括，但不限于，视网膜色素变性（也称为色素性视网膜炎）、视锥细胞-视杆细胞营养不良、先天性静止性夜盲、年龄相关性黄斑变性、斯特格病、利伯先天性黑矇、贝斯特卵黄状黄斑营养不良、黄斑中心营养不良、Bietti晶体角膜视网膜营养不良、尤塞氏综合症等。Specifically, the retinal degeneration-related diseases include, but are not limited to, retinitis pigmentosa (also known as retinitis pigmentosa), cone-rod dystrophy, congenital stationary night blindness, age-related macular degeneration, Stargardt's disease, Leber congenital amaurosis, Best's vitelliform macular dystrophy, central macular dystrophy, Bietti crystalline corneal retinal dystrophy, Usher's syndrome, etc.
具体地，所述视网膜变性可衍生自白内障、糖尿病或者青光眼等。Specifically, the retinal degeneration may be derived from cataracts, diabetes, glaucoma, or the like.
具体地，所述药物可以采用任何合适的给药方式，特别是眼部给药方式（例如眼内给药、眼表给药），例如滴眼液给药、眼膏给药、结膜下注射给药、玻璃体腔注射给药、球后注射，特别是玻璃体腔注射给药。Specifically, the drug can be administered in any suitable manner, in particular, in ocular administration (e.g., intraocular administration, ocular surface administration), such as eye drops, eye ointment, subconjunctival injection, vitreous cavity injection, retrobulbar injection, in particular, vitreous cavity injection.
具体地，所述药物可以被配制为任何合适的制剂形式，例如，但不限于，霜、泡沫、膏、软膏、乳剂、液体溶液、滴眼剂、注射剂、粉针剂、凝胶、喷雾、悬浮液、微乳液、眼罩或隐形眼镜等，特别是注射剂。Specifically, the drug can be formulated into any suitable preparation form, such as, but not limited to, cream, foam, cream, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, etc., especially injection.
具体地，所述药物（或所述细胞内纳米囊泡）用于以下中的一项或多项：Specifically, the drug (or the intracellular nanovesicle) is used for one or more of the following:
（1）视网膜损伤修复；(1) Retinal damage repair;
（2）角膜损伤修复。(2) Corneal damage repair.
更具体地，所述药物（或所述细胞内纳米囊泡）用于以下中的一项或多项：More specifically, the drug (or the intracellular nanovesicle) is used for one or more of the following:
（1a）保护视网膜结构（减缓视网膜变薄）；(1a) Protect retinal structure (slow down retinal thinning);
（1b）保护视网膜电生理功能；(1b) Protecting retinal electrophysiological function;
（1c）减少视网膜细胞凋亡；(1c) Reduce retinal cell apoptosis;
（1d）减少视网膜光感受器细胞丢失、细胞死亡；(1d) Reduce retinal photoreceptor cell loss and cell death;
（1e）减少视网膜炎症；(1e) Reduce retinal inflammation;
（1f）抑制视网膜内质网应激；(1f) inhibit retinal endoplasmic reticulum stress;
（2a）促进角膜上皮细胞增殖、趋化、迁移；(2a) Promote corneal epithelial cell proliferation, chemotaxis, and migration;
（2b）促进角膜上皮、基质修复；(2b) Promote the repair of corneal epithelium and matrix;
（2c）促进角膜敏感性恢复；(2c) Promote the recovery of corneal sensitivity;
（2d）减少角膜新生血管形成。(2d) Reduce corneal neovascularization.
在本发明的一些实施方案中，所述药物（或所述细胞内纳米囊泡）用于实现（1a）至（1f）。In some embodiments of the present invention, the drug (or the intracellular nanovesicle) is used to achieve (1a) to (1f).
在本发明的一些实施方案中，所述药物（或所述细胞内纳米囊泡）用于实现（2a）至（2d）。In some embodiments of the present invention, the drug (or the intracellular nanovesicle) is used to achieve (2a) to (2d).
本发明的第二方面，提供一种成体干细胞来源的细胞内纳米囊泡在制备角膜损伤修复或视网膜损伤修复的药物中的应用。The second aspect of the present invention provides a use of intracellular nanovesicles derived from adult stem cells in the preparation of a drug for repairing corneal damage or retinal damage.
具体地，所述成体干细胞来源的细胞内纳米囊泡如本发明第一方面所述。Specifically, the intracellular nanovesicles derived from adult stem cells are as described in the first aspect of the present invention.
具体地，所述角膜损伤可以是各种原因（如外力、高压气体或液体冲击、化学刺激、感染、角膜接触镜佩戴不当、营养不良、角膜肿瘤等）引起的角膜结构破坏，例如机械性角膜损伤、化学性角膜损伤（如角膜碱烧伤）等，其表现可包括，但不限于，角膜上皮损伤、角膜基质层水肿、角膜破裂、角膜知觉减退、角膜变薄、角膜脱离、角膜劈裂、角膜震荡、角膜的出血、渗血或者是缺血等。在本发明的一个实施例中，所述角膜损伤为机械性角膜损伤。在本发明另一个实施例中，所述角膜损伤为角膜碱烧伤。具体地，所述视网膜损伤可以是各种原因引起的视网膜损伤，例如光化学损伤，其表现可包括，但不限于，视网膜变薄、视网膜脱离、视网膜劈裂、视网膜震荡、视网膜的出血、渗出或者是缺血等。Specifically, the corneal injury may be corneal structural damage caused by various reasons (such as external force, high-pressure gas or liquid impact, chemical stimulation, infection, improper wearing of corneal contact lenses, malnutrition, corneal tumors, etc.), such as mechanical corneal injury, chemical corneal injury (such as corneal alkali burns), etc., and its manifestations may include, but are not limited to, corneal epithelial injury, corneal stroma edema, corneal rupture, corneal hypoesthesia, corneal thinning, corneal detachment, corneal splitting, corneal concussion, corneal bleeding, exudation or ischemia, etc. In one embodiment of the present invention, the corneal injury is mechanical corneal injury. In another embodiment of the present invention, the corneal injury is corneal alkali burns. Specifically, the retinal injury may be retinal injury caused by various reasons, such as photochemical injury, and its manifestations may include, but are not limited to, retinal thinning, retinal detachment, retinal splitting, retinal concussion, retinal bleeding, exudation or ischemia, etc.
本发明的第三方面，提供一种预防和/或治疗眼科疾病的方法，其包括向有此需要的受试者给药成体干细胞来源的细胞内纳米囊泡（如本发明第一方面所述）的步骤。The third aspect of the present invention provides a method for preventing and/or treating ophthalmic diseases, comprising the step of administering adult stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.
具体地，所述疾病如本发明第一方面所述。Specifically, the disease is as described in the first aspect of the present invention.
具体地，所述受试者为哺乳动物，特别是人类。Specifically, the subject is a mammal, especially a human.
具体地，所述给药可以采用任何合适的给药方式，特别是眼部给药方式（例如眼内给药、眼表给药、眼周给药），例如玻璃体腔注射给药、前房注射、玻璃体植入给药；滴眼液给药、眼膏给药、眼凝胶给药；结膜下注射给药、球后注射、球周给药、球筋膜下给药等，特别是玻璃体腔注射给药。Specifically, the administration can be carried out by any suitable administration method, in particular, ocular administration methods (such as intraocular administration, ocular surface administration, periocular administration), such as intravitreal injection, anterior chamber injection, vitreous implantation; eye drops, eye ointment, eye gel; subconjunctival injection, retrobulbar injection, peribulbar injection, subbulbar administration, etc., in particular intravitreal injection.
具体地，所述给药量取决于多种因素，包括受试者的年龄、体重、性别、病症、严重程度、给药途径和频率等，因此可以有多种变化；在本发明的一些实施方案中，所述给药量可以为1μg-60μg囊泡/眼，例如，1μg囊泡/眼、2μg囊泡/眼、4μg囊泡/眼、5μg囊泡/眼、6μg囊泡/眼、8μg囊泡/眼、10μg囊泡/眼、15μg囊泡/眼、20μg囊泡/眼、25μg囊泡/眼、30μg囊泡/眼、35μg囊泡/眼、40μg囊泡/眼、45μg囊泡/眼、50μg囊泡/眼、55μg囊泡/眼、60μg囊泡/眼。Specifically, the dosage depends on many factors, including the age, weight, sex, disease, severity, route and frequency of administration of the subject, and therefore may vary. In some embodiments of the present invention, the dosage may be 1 μg-60 μg vesicle/eye, for example, 1 μg vesicle/eye, 2 μg vesicle/eye, 4 μg vesicle/eye, 5 μg vesicle/eye, 6 μg vesicle/eye, 8 μg vesicle/eye, 10 μg vesicle/eye, 15 μg vesicle/eye, 20 μg vesicle/eye, 25 μg vesicle/eye, 30 μg vesicle/eye, 35 μg vesicle/eye, 40 μg vesicle/eye, 45 μg vesicle/eye, 50 μg vesicle/eye, 55 μg vesicle/eye, 60 μg vesicle/eye.
在本发明的一些实施例中，所述给药方式为玻璃体腔注射给药。In some embodiments of the present invention, the administration method is intravitreal injection.
在本发明的一些实施例中，所述给药方式为眼表给药，如滴眼液给药。In some embodiments of the present invention, the administration method is ocular surface administration, such as eye drop administration.
本发明提供的小细胞内纳米囊泡，相对于以外泌体为主要成分的小细胞外囊泡，粒径较小，粒径分布范围窄，在不同温度下更稳定，具有良好的组织相容性，在玻璃体腔注药方式下分布的范围和程度更广，其作为载体负载药物时具有更高的包封率和载药率，例如在玻璃体腔注射形式下负载的药物可更快被吸收，在医药领域（特别是眼部疾病治疗）中具有非常好的应用和研究价值。Compared with small extracellular vesicles with exosomes as the main component, the small intracellular nanovesicles provided by the present invention have smaller particle size, narrower particle size distribution range, are more stable at different temperatures, have good tissue compatibility, and have a wider range and degree of distribution under the vitreous cavity injection method. When used as a carrier to load drugs, it has a higher encapsulation rate and drug loading rate. For example, the loaded drugs in the form of vitreous cavity injection can be absorbed faster, and have very good application and research value in the medical field (especially in the treatment of eye diseases).
本发明的MSC-sIVs能减缓视网膜变薄，在保护视网膜光感受器细胞方面表现出更优异的疗效。MSC-sIVs能够负调控内质网应激，对内质网应激信号的抑制效果显著，并能够有效减少视网膜凋亡。总之，本发明的MSC-sIVs对于蓝光诱导的视网膜光损伤和rd10视网膜色素变性均具有明显的保护作用。The MSC-sIVs of the present invention can slow down retinal thinning and show a more excellent therapeutic effect in protecting retinal photoreceptor cells. MSC-sIVs can negatively regulate endoplasmic reticulum stress, have a significant inhibitory effect on endoplasmic reticulum stress signals, and can effectively reduce retinal apoptosis. In short, the MSC-sIVs of the present invention have a significant protective effect on blue light-induced retinal light damage and rd10 retinitis pigmentosa.
本发明的MSC-sIVs能改善因创伤等导致的角膜损伤，减轻角膜混浊、结膜充血、结膜水肿程度，减少角膜新生血管的生成。The MSC-sIVs of the present invention can improve corneal damage caused by trauma and the like, reduce corneal opacity, conjunctival congestion, conjunctival edema, and reduce the generation of corneal neovascularization.
本发明的间充质干细胞来源的小细胞内纳米囊泡在医药领域（特别是，角膜损伤、视网膜损伤和变性）中具有非常好的应用和研究价值。The small intracellular nanovesicles derived from mesenchymal stem cells of the present invention have very good application and research value in the medical field (especially, corneal damage, retinal damage and degeneration).
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1所示为细胞内纳米囊泡的生产方法的流程和步骤示意图。其中图1A示流程图，图1B示步骤。Figure 1 shows a schematic diagram of the process and steps of the method for producing intracellular nanovesicles, wherein Figure 1A shows a flow chart, and Figure 1B shows the steps.
图2所示为对细胞内纳米囊泡的分离参数的优化过程。其中，图2A-2B所示分别为在20%的超声振幅，不同作用时间下所得sIVs的蛋白质产量（图2A）和囊泡产量（图2B）；在20%的超声振幅下，作用时间超过低于10秒或高于20秒，囊泡产量骤降。图2C-2D所示分别为在15s的超声作用时间，不同超声振幅下所得sIVs的蛋白质产量（图2C）和囊泡产量（图2D）；在15s的超声时间下，超声振幅高于25%，囊泡产量骤降。图2E所示为在15s的超声作用时间，不同超声振幅下所得sIVs的透射电镜图，比例尺：100nm。图2F所示为在20%的超声振幅，不同作用时间下所得sIVs的透射电镜图，比例尺：100nm。Figure 2 shows the optimization process of the separation parameters of intracellular nanovesicles. Among them, Figures 2A-2B show the protein yield (Figure 2A) and vesicle yield (Figure 2B) of sIVs obtained at 20% ultrasonic amplitude and different action times; at 20% ultrasonic amplitude, the action time is less than 10 seconds or more than 20 seconds, and the vesicle yield drops sharply. Figures 2C-2D show the protein yield (Figure 2C) and vesicle yield (Figure 2D) of sIVs obtained at 15s ultrasonic action time and different ultrasonic amplitudes; at 15s ultrasonic time, the ultrasonic amplitude is higher than 25%, and the vesicle yield drops sharply. Figure 2E shows the transmission electron micrographs of sIVs obtained at 15s ultrasonic action time and different ultrasonic amplitudes, scale: 100nm. Figure 2F shows the transmission electron micrographs of sIVs obtained at 20% ultrasonic amplitude and different action times, scale: 100nm.
图3所示为MSCs细胞的sEVs和sIVs的透射电镜图。宽视野比例尺：200nm；特写比例尺：200nm。Figure 3 shows transmission electron microscopy images of sEVs and sIVs of MSCs cells. Wide field scale bar: 200 nm; close-up scale bar: 200 nm.
图4所示为纳米粒度分析结果，其显示MSCs细胞及其sEVs和sIVs的粒径分布。FIG4 shows the results of nanoparticle size analysis, which shows the particle size distribution of MSCs cells and their sEVs and sIVs.
图5所示为MSCs细胞及其sEVs和sIVs粒径的统计学分析结果（p＜0.01，p＜0.001表示各组间显著性差异）。Figure 5 shows the statistical analysis results of the particle sizes of MSCs cells, sEVs and sIVs ( p＜0.01, p＜0.001 indicates significant difference among the groups).
图6所示为等细胞个数下MSCs细胞的sEVs和sIVs囊泡个数（A）和总蛋白产量的统计学分析结果（B）（p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。Figure 6 shows the statistical analysis results of the number of sEVs and sIVs vesicles (A) and total protein production of MSCs cells under equal cell numbers (B) p＜0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).
图7所示为考马斯亮蓝染色结果，其显示MSCs细胞及其sEVs和sIVs的蛋白分布。FIG. 7 shows the results of Coomassie Brilliant Blue staining, which shows the protein distribution of MSCs cells and their sEVs and sIVs.
图8所示为Western blot结果，其显示MSCs细胞的外泌体标志蛋白（Alix, HSP70,TSG101, CD63, CD81）表达情况。FIG8 shows the results of Western blot, which shows the expression of exosome marker proteins (Alix, HSP70, TSG101, CD63, CD81) of MSCs cells.
图9所示为不同温度下MSCs的sEVs和sIVs的透射电镜图。比例尺：200nm。Figure 9 shows transmission electron microscopy images of sEVs and sIVs of MSCs at different temperatures. Scale bar: 200 nm.
图10所示为不同温度（-80℃, 4℃ 和37℃）下MSCs细胞的sEVs和sIVs的纳米粒度分析结果。其中图10A显示粒径分布，图10B显示粒径统计分析结果（p＜0.05，p＜0.01表示各组间显著性差异）。Figure 10 shows the nanoparticle size analysis results of sEVs and sIVs of MSCs cells at different temperatures (-80°C, 4°C and 37°C). Figure 10A shows the particle size distribution, and Figure 10B shows the particle size statistical analysis results ( p＜0.05, p＜0.01 indicates significant difference among the groups).
图11所示为在MSCs细胞中的sIVs特有蛋白，丰度由高到低排列，展示各自的最高表达的前50个蛋白。FIG. 11 shows the sIVs-specific proteins in MSCs cells, arranged from high to low abundance, showing the top 50 proteins with the highest expression.
图12所示为超分辨显微镜与全内反射荧光结构照明显微镜检测结果。其中，图12A显示TIRF-SIM模式（示细胞膜），其显示细胞膜表面存在CD63阳性（绿色）区域，而几乎没有观察到TMEM214阳性（红色）区域。图12B显示宽场-2DSM模式（示全细胞），其证明在整个细胞中同时存在CD63阳性和TMEM214阳性信号。图12C显示动态观察活细胞的分时段截图，左侧绿色为CD63标记的细胞内晚期内体、sEVs和细胞膜，0s箭头所示sEVs刚被细胞膜释放至细胞外，6min至14min逐渐远离细胞膜，右侧红色为TMEM214，显示sIVs在细胞内弥散分布，未释放到细胞外。Figure 12 shows the results of super-resolution microscopy and total internal reflection fluorescence structured illumination microscopy. Among them, Figure 12A shows the TIRF-SIM mode (showing the cell membrane), which shows that there are CD63-positive (green) areas on the cell membrane surface, while almost no TMEM214-positive (red) areas are observed. Figure 12B shows the widefield-2DSM mode (showing the whole cell), which proves that CD63-positive and TMEM214-positive signals exist simultaneously in the whole cell. Figure 12C shows a time-division screenshot of the dynamic observation of living cells. The green on the left is the CD63-labeled intracellular late endosomes, sEVs and cell membranes. The arrow at 0s shows that sEVs have just been released from the cell membrane to the outside of the cell, and gradually move away from the cell membrane from 6min to 14min. The red on the right is TMEM214, showing that sIVs are diffusely distributed in the cell and are not released outside the cell.
图13所示为韦恩图，其显示MSCs细胞、sEVs和sIVs中总蛋白质种类。FIG. 13 is a Venn diagram showing the total protein species in MSCs cells, sEVs, and sIVs.
图14所示为MSCs细胞、sEVs和sIVs中鉴定的总蛋白质的主成分分析结果。FIG. 14 shows the principal component analysis results of the total proteins identified in MSCs cells, sEVs and sIVs.
图15所示为热图，其显示MSCs细胞的sEVs和sIVs之间存在差异表达蛋白。FIG. 15 is a heat map showing the presence of differentially expressed proteins between sEVs and sIVs of MSCs cells.
图16所示为火山图，其显示MSCs细胞的sEVs和sIVs之间的前五个显著差异表达蛋白。FIG. 16 shows a volcano plot showing the top five significantly differentially expressed proteins between sEVs and sIVs of MSCs cells.
图17所示为热图，其显示MSCs细胞的sEVs和sIVs之间的外泌体标记物的差异表达。FIG. 17 is a heat map showing the differential expression of exosomal markers between sEVs and sIVs of MSCs cells.
图18所示为热图，其显示MSCs细胞的sEVs和sIVs之间的细胞器标记物的差异表达。FIG. 18 shows a heat map showing the differential expression of organelle markers between sEVs and sIVs of MSCs cells.
图19所示为热图，其显示MSCs细胞的sEVs和sIVs之间的Clathrin家族蛋白的差异表达。FIG. 19 is a heat map showing the differential expression of Clathrin family proteins between sEVs and sIVs of MSCs cells.
图20所示为MSCs的sIVs表达蛋白的细胞成分富集分析。FIG. 20 shows the cellular component enrichment analysis of sIVs-expressed proteins in MSCs.
图21所示为MSCs的sIVs表达蛋白的生物进程富集分析。FIG. 21 shows the biological process enrichment analysis of sIVs-expressed proteins in MSCs.
图22所示为蛋白质谱检测的MSCs的sEVs和sIVs携带的IL-1β和IGF2的细胞因子水平差异。其中，图22A所示为蛋白质谱检测的MSCs的sEVs和sIVs携带的IL-1β的细胞因子水平差异，图22B所示为蛋白质谱检测的MSCs的sEVs和sIVs携带的IGF2的细胞因子水平差异（p＜0.05，p＜0.001表示各组间显著性差异）。FIG22 shows the differences in cytokine levels of IL-1β and IGF2 carried by sEVs and sIVs of MSCs detected by protein profiling. FIG22A shows the differences in cytokine levels of IL-1β carried by sEVs and sIVs of MSCs detected by protein profiling, and FIG22B shows the differences in cytokine levels of IGF2 carried by sEVs and sIVs of MSCs detected by protein profiling ( p＜0.05, p＜0.001 indicates significant difference among the groups).
图23所示为ELISA检测的MSCs的sEVs和sIVs携带的IGF-1、EGF、IL-10、IL-6和TNFα的细胞因子水平差异。其中，图23A所示为ELISA检测的MSCs的sEVs和sIVs携带的IGF-1的细胞因子水平差异，图23B所示为ELISA检测的MSCs的sEVs和sIVs携带的EGF的细胞因子水平差异，图23C所示为ELISA检测的MSCs的sEVs和sIVs携带的IL-10的细胞因子水平差异，图23D所示为ELISA检测的MSCs的sEVs和sIVs携带的IL-6的细胞因子水平差异，图23E所示为ELISA检测的MSCs的sEVs和sIVs携带的TNFα的细胞因子水平差异（p＜0.01，p＜0.001表示各组间显著性差异，ns示无统计学差异）。Figure 23 shows the differences in the levels of IGF-1, EGF, IL-10, IL-6 and TNFα carried by sEVs and sIVs of MSCs detected by ELISA. Among them, Figure 23A shows the differences in the levels of IGF-1 carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23B shows the differences in the levels of EGF carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23C shows the differences in the levels of IL-10 carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23D shows the differences in the levels of IL-6 carried by sEVs and sIVs of MSCs detected by ELISA, and Figure 23E shows the differences in the levels of TNFα carried by sEVs and sIVs of MSCs detected by ELISA ( p＜0.01, p<0.001 indicates significant difference among the groups, ns indicates no statistical difference).
图24所示为MSCs细胞的sEV和sIVs中的相对RNA丰度（ns示无统计学差异）。FIG. 24 shows the relative RNA abundance in sEVs and sIVs of MSCs cells (ns indicates no statistical difference).
图25所示为来自MSCs细胞的sEVs和sIVs的小RNA读取的小非编码RNA的百分比。miRNA：micro-RNA；snoRNA：小核仁RNA；snRNA：小核RNA；tRNA：转移RNA；rRNA：核糖体RNA。Figure 25 shows the percentage of small non-coding RNAs read from small RNAs of sEVs and sIVs from MSCs cells. miRNA: micro-RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; tRNA: transfer RNA; rRNA: ribosomal RNA.
图26所示为韦恩图，其显示MSCs细胞的sEVs和sIVs含有的miRNA种类。FIG. 26 is a Venn diagram showing the types of miRNAs contained in sEVs and sIVs of MSCs cells.
图27所示为MSCs细胞miRNA数据集的主成分分析结果。FIG. 27 shows the principal component analysis results of the MSCs cell miRNA dataset.
图28所示为火柴图，其显示了MSCs细胞的sEVs和sIVs中前10个高丰度miRNA。FIG. 28 is a matchstick chart showing the top 10 highly abundant miRNAs in sEVs and sIVs of MSCs cells.
图29所示为热图，其显示MSCs细胞的sEVs和sIVs之间存在较多差异表达的miRNA。FIG. 29 is a heat map showing that there are many differentially expressed miRNAs between sEVs and sIVs of MSCs cells.
图30所示为火山图，其显示MSCs细胞的sEVs和sIVs之间差异表达的前5个miRNA。横坐标表示miRNA在不同样本或比较组合间的表达倍数变化（log2差异倍数），纵坐标表示表达差异的显著性水平。Figure 30 shows a volcano plot showing the top 5 differentially expressed miRNAs between sEVs and sIVs of MSCs cells. The abscissa represents the expression fold change (log2 difference fold) of miRNA between different samples or comparison combinations, and the ordinate represents the significance level of the expression difference.
图31所示MSCs来源的sEVs和sIVs中差异表达的miRNA候选靶基因的富集分析。图31A示GO分析，生物过程（Biological Process, BP）、细胞组分(Cellular Component ,CC）和分子功能（Molecular Function, MF）的前10个条目。图31B示MSCs的sEVs和sIVs中差异表达的miRNA候选靶基因的KEGG富集分析。Figure 31 shows the enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs derived from MSCs. Figure 31A shows the top 10 entries of GO analysis, biological process (BP), cellular component (CC) and molecular function (MF). Figure 31B shows the KEGG enrichment analysis of differentially expressed miRNA candidate target genes in sEVs and sIVs of MSCs.
图32所示为MSCs细胞的sEVs和sIVs所含代谢物种类及占比。FIG32 shows the types and proportions of metabolites contained in sEVs and sIVs of MSCs cells.
图33所示为MSCs细胞sEVs和sIVs所含脂质的主成分分析结果。FIG. 33 shows the principal component analysis results of lipids contained in MSCs cell sEVs and sIVs.
图34所示为热图，其显示MSCs细胞sEVs和sIVs所含差异脂质类型。FIG. 34 is a heat map showing the differential lipid types contained in MSCs cell sEVs and sIVs.
图35所示为MSCs细胞sIVs组对sEVs组的脂质组柱形图。图的横坐标代表该组对比各物质含量的相对变化百分比。如果含量相对变化百分比为零，表示该物质在两组中的含量相同；含量相对变化百分比为正数，表示该物质在sIVs组中的含量更高；含量相对变化百分比为负数，表示该物质在sEVs组中的含量更高。脂质组柱形图的纵坐标表示脂质的分类信息。Figure 35 shows a lipidome bar graph of the MSCs cell sIVs group versus the sEVs group. The horizontal axis of the graph represents the relative percentage change in the content of each substance in the group. If the relative percentage change in content is zero, it means that the content of the substance in the two groups is the same; if the relative percentage change in content is a positive number, it means that the content of the substance in the sIVs group is higher; if the relative percentage change in content is a negative number, it means that the content of the substance in the sEVs group is higher. The vertical axis of the lipidome bar graph represents the classification information of lipids.
图36所示为细胞内纳米囊泡被体外培养的RPE细胞内吞的能力的考察结果。其中，图36A和B所示为DiD标记的sEVs和sIVs与RPE细胞共孵育3h、12h、24h和48h。绿色为细胞骨架，红色为囊泡，DAPI染色显示蓝色为细胞核。比例尺：20μm。图36C所示为细胞内的DiD荧光强度统计学结果（p＜0.05、p＜0.01和p＜0.001表示各组间显著性差异）。FIG36 shows the results of investigating the ability of intracellular nanovesicles to be internalized by RPE cells cultured in vitro. FIG36A and FIG36B show that DiD-labeled sEVs and sIVs were co-incubated with RPE cells for 3 h, 12 h, 24 h, and 48 h. Green is the cytoskeleton, red is the vesicle, and DAPI staining shows that blue is the cell nucleus. Scale bar: 20 μm. FIG36C shows the statistical results of DiD fluorescence intensity in the cell ( p＜0.05, p<0.01 and p＜0.001 indicates significant difference among the groups).
图37所示为细胞内纳米囊泡被体外培养的HRMECs细胞内吞的能力的考察结果。其中，图37A和B所示为DiD标记的sEVs和sIVs与HRMECs共孵育3h、12h、24h和48h。绿色为细胞骨架，红色为囊泡，DAPI染色显示蓝色为细胞核。比例尺：20μm。图37C所示为细胞内的DiD荧光强度统计学结果（p＜0.05、p＜0.01和p＜0.001表示各组间显著性差异）。FIG37 shows the results of investigating the ability of intracellular nanovesicles to be internalized by HRMECs cultured in vitro. FIG37A and FIG37B show DiD-labeled sEVs and sIVs co-incubated with HRMECs for 3 h, 12 h, 24 h, and 48 h. Green is the cytoskeleton, red is the vesicle, and DAPI staining shows that blue is the cell nucleus. Scale bar: 20 μm. FIG37C shows the statistical results of DiD fluorescence intensity in cells ( p＜0.05, p<0.01 and p＜0.001 indicates significant difference among the groups).
图38所示为细胞内纳米囊泡被视网膜内吞的能力的考察结果。其中，图38A示DiD标记的sEVs和sIVs经结膜下注射24h和48h，在视网膜切片上的分布。图38B所示为DiD标记的sEVs和sIVs经玻璃体腔注射8h和24h，在视网膜切片上的分布。DAPI染色显示细胞核。比例尺：20μm。图38C所示为结膜下注射组视网膜内的DiD荧光强度统计学结果。图38D所示为玻璃体腔注射组视网膜内的DiD荧光强度统计学结果（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。Figure 38 shows the results of investigating the ability of intracellular nanovesicles to be internalized by the retina. Figure 38A shows the distribution of DiD-labeled sEVs and sIVs on retinal sections 24h and 48h after subconjunctival injection. Figure 38B shows the distribution of DiD-labeled sEVs and sIVs on retinal sections 8h and 24h after intravitreal injection. DAPI staining shows the cell nucleus. Scale bar: 20μm. Figure 38C shows the statistical results of DiD fluorescence intensity in the retina of the subconjunctival injection group. Figure 38D shows the statistical results of DiD fluorescence intensity in the retina of the intravitreal injection group ( p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).
图39所示为MSC-sIVs保护视网膜厚度免受蓝光损伤。其中，图39A所示为代表性热图显示了视网膜的厚度。图39B所示为视网膜的代表性SD-OCT扫描。图39C所示为视网膜的平均厚度统计学结果，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间统计学差异）。FIG39 shows that MSC-sIVs protect retinal thickness from blue light damage. FIG39A shows a representative heat map showing retinal thickness. FIG39B shows a representative SD-OCT scan of the retina. FIG39C shows the statistical results of the average thickness of the retina, where BL represents light damage model mice, L is a low-dose administration group, and H is a high-dose administration group ( p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates statistically significant difference between the groups).
图40所示为MSC-sIVs保护视网膜外核层细胞免受蓝光诱导的损伤。其中，图40A所示为各组H&E染色的代表性视网膜图像（GCL：神经节细胞层；INL：核内层；ONL：外核层）。黑色比例尺：100μm；白色比例尺：20μm。图40B所示为蓝光诱导一周后，在视网膜切片的八个区域中计数ONL细胞核，从距视神经头250μm开始，沿背侧/上缘（横坐标上的正值）和腹侧周边/下缘（横坐标上的负值）每250μm或500μm向外延伸一次，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（与BL-PBS相比，每组之间的统计差异用相应的颜色表示，p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组之间的统计学差异）。Figure 40 shows that MSC-sIVs protect retinal outer nuclear layer cells from blue light-induced damage. Figure 40A shows representative retinal images of H&E staining of each group (GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). Black scale bar: 100μm; white scale bar: 20μm. Figure 40B shows the counting of ONL nuclei in eight areas of retinal sections one week after blue light induction, starting from 250μm from the optic nerve head and extending outward every 250μm or 500μm along the dorsal/upper edge (positive values on the horizontal axis) and ventral periphery/lower edge (negative values on the horizontal axis), where BL represents light damage model mice, L is the low-dose administration group, and H is the high-dose administration group (compared with BL-PBS, the statistical differences between each group are represented by the corresponding colors, p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates statistically significant difference between the groups).
图41所示为MSC-sIVs保护视网膜电生理功能免受蓝光损伤。其中，图41A所示为刺激闪光下来自每个治疗组的单个小鼠的一只眼睛的代表性ERG波形[2.2Log（cd.s/m2）]。图41B所示为各治疗组ERG a波和b波的平均振幅，其中BL代表光损伤模型小鼠（与BL-PBS相比，各组之间的统计差异用相应的颜色表示；p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组之间的统计学差异）。FIG41 shows that MSC-sIVs protect retinal electrophysiological function from blue light damage. FIG41A shows a representative ERG waveform [2.2Log (cd.s/m2)] of one eye from a single mouse in each treatment group under the stimulus flash. FIG41B shows the average amplitude of ERG a-wave and b-wave in each treatment group, where BL represents light damage model mice (compared with BL-PBS, the statistical differences between groups are indicated by corresponding colors; p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates statistically significant difference between the groups).
图42所示为MSC-sIVs减少蓝光诱导的视网膜凋亡。其中，图42A所示为各组具有代表性的形态学图像；绿色信号是TUNEL阳性，蓝色信号是DAPI染色。比例尺：20μm。图42B所示为从整个视网膜计数的TUNEL阳性细胞核，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（p<0.01，p<0.0001表示与NoBL-PBS组的统计学差异。####p<0.0001表示与BL-PBS组的统计学差异）。FIG42 shows that MSC-sIVs reduce blue light-induced retinal apoptosis. FIG42A shows representative morphological images of each group; green signals are TUNEL positive, and blue signals are DAPI staining. Scale bar: 20 μm. FIG42B shows TUNEL positive cell nuclei counted from the entire retina, where BL represents light damage model mice, L is a low-dose administration group, and H is a high-dose administration group ( p<0.01, p<0.0001 indicates statistical difference compared with NoBL-PBS group.#### p<0.0001 indicates statistical difference compared with BL-PBS group).
图43所示为MSC-sIVs减少蓝光诱导的光感受器细胞损伤。其中，图43A所示为用免疫荧光法测定小鼠视网膜中视紫红质蛋白的表达。细胞核用DAPI（蓝色）染色。比例尺：20μm。图43B所示为视网膜中视紫红质蛋白的相对表达，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（p<0.05、p<0.01、p<0.001和p<0.0001表示与NoBL-PBS组相比的统计学差异；##p<0.01，####p<0.0001表示与BL-PBS组相比的统计学差异；&p<0.05，&&&&p<0.0001表示同剂量sEVs与sIVs之间的统计学差异）。FIG43 shows that MSC-sIVs reduce blue light-induced photoreceptor cell damage. FIG43A shows the expression of rhodopsin protein in the mouse retina measured by immunofluorescence. The nucleus was stained with DAPI (blue). Scale bar: 20 μm. FIG43B shows the relative expression of rhodopsin protein in the retina, where BL represents light-damaged model mice, L is a low-dose administration group, and H is a high-dose administration group ( p<0.05, p<0.01, p<0.001 and p<0.0001 indicates statistical difference compared with the NoBL-PBS group;## p<0.01,#### p<0.0001 indicate statistical difference compared with the BL-PBS group;& p<0.05,&&&& p<0.0001 indicate statistical difference between sEVs and sIVs of the same dose).
图44所示为视网膜中GFAP和视紫红质的蛋白质印迹分析。其中，图44A所示为小鼠视网膜中GFAP和视紫红质的蛋白质印迹分析。图44B所示为定量分析，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（p<0.05、p<0.01、p<0.001和p<0.0001表示与NoBL-PBS组相比的统计学差异，#p<0.05和##p<0.01表示与BL-PBS组相比的统计学差异，&p<0.05，&&p<0.01表示同剂量sEVs与sIVs之间的统计学差异）。FIG44 shows the Western blot analysis of GFAP and rhodopsin in the retina. FIG44A shows the Western blot analysis of GFAP and rhodopsin in the mouse retina. FIG44B shows the quantitative analysis, where BL represents the light damage model mouse, L is the low-dose administration group, and H is the high-dose administration group ( p<0.05, p<0.01, p<0.001 and p<0.0001 indicates statistical difference compared with the NoBL-PBS group,# p<0.05 and## p<0.01 indicate statistical difference compared with the BL-PBS group,& p<0.05,&& p<0.01 indicate statistical difference between sEVs and sIVs at the same dose).
图45所示为MSC-sIVs抑制蓝光诱导的视网膜Müller细胞活化。其中，图45A所示为免疫荧光法用于测定小鼠视网膜中GFAP的表达。细胞核用DAPI（蓝色）染色。比例尺：20μm。图45B所示为视网膜中GFAP的相对表达，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（p<0.05、p<0.01、p<0.001和p<0.0001表示与NoBL-PBS组相比的统计学差异，#p<0.05和##p<0.01表示与BL-PBS组相比的统计学差异）。FIG45 shows that MSC-sIVs inhibit blue light-induced retinal Müller cell activation. FIG45A shows that immunofluorescence was used to determine the expression of GFAP in the mouse retina. The nuclei were stained with DAPI (blue). Scale bar: 20 μm. FIG45B shows the relative expression of GFAP in the retina, where BL represents light-damaged model mice, L is a low-dose administration group, and H is a high-dose administration group ( p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference compared with the NoBL-PBS group,# p<0.05 and## p<0.01 indicate a statistically significant difference compared with the BL-PBS group).
图46所示为MSC-sIVs抑制蓝光诱导的视网膜小胶质细胞数量增加。其中，图46A所示为Iba1阳性细胞在视网膜中的分布。比例尺：20μm。图46B所示为整个视网膜中Iba1阳性细胞的定量分析。图46C所示为内层视网膜Iba1阳性细胞的定量分析。图46D所示为外层视网膜Iba1阳性细胞的定量分析，其中BL代表光损伤模型小鼠，L为低剂量给药组，H为高剂量给药组（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表明与NoBL-PBS组存在显著差异，#p<0.05、##p<0.01、###p<0.001和####p<0.0001表明与BL-PBS组存在显著差异，&p＜0.05和&&p＜0.01表明在相同剂量下组间存在显著差异）。FIG46 shows that MSC-sIVs inhibit the increase in the number of retinal microglia induced by blue light. FIG46A shows the distribution of Iba1-positive cells in the retina. Scale bar: 20 μm. FIG46B shows the quantitative analysis of Iba1-positive cells in the entire retina. FIG46C shows the quantitative analysis of Iba1-positive cells in the inner retina. FIG46D shows the quantitative analysis of Iba1-positive cells in the outer retina, where BL represents light-damaged model mice, L is a low-dose administration group, and H is a high-dose administration group ( p＜0.05, p＜0.01, p<0.001 and p＜0.0001 indicated a significant difference from the NoBL-PBS group,# p<0.05,## p<0.01,### p<0.001 and#### p<0.0001 indicated a significant difference from the BL-PBS group,& p＜0.05 and&& p＜0.01 indicated significant differences between the groups at the same dose).
图47所示为MSC-sIVs减缓rd10小鼠外核层细胞丢失。图47A所示为各组H&E染色的代表性视网膜图像（GCL：神经节细胞层；INL：核内层；ONL：核外层）。黑色比例尺：100μm；白色比例尺：20μm。图47B所示为在视网膜切片的八个离散区域中计数ONL的细胞核，从距视神经头250μm开始，沿着背侧/上缘（横坐标上的正值）和腹侧周边/下缘（横坐标上的负值）每250μm或500μm向外延伸一次（与rd10-PBS相比，每组之间的统计差异用相应的颜色表示，粉色表示rd10-sEVs与rd10-sIVs之间的比较，p＜0.05、p＜0.01和p＜0.0001表示各组之间的显著差异）。Figure 47 shows that MSC-sIVs slowed the loss of outer nuclear layer cells in rd10 mice. Figure 47A shows representative retinal images of each group stained with H&E (GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer). Black scale bar: 100 μm; white scale bar: 20 μm. Figure 47B shows the counting of cell nuclei in the ONL in eight discrete areas of retinal sections, starting from 250 μm from the optic nerve head and extending outward every 250 μm or 500 μm along the dorsal/superior edge (positive values on the abscissa) and ventral periphery/inferior edge (negative values on the abscissa) (compared with rd10-PBS, the statistical differences between each group are indicated by the corresponding color, pink indicates the comparison between rd10-sEVs and rd10-sIVs, p＜0.05, p<0.01 and p<0.0001 indicates significant difference between the groups).
图48所示为MSC-sIVs挽救rd10小鼠视网膜电生理功能。图48A所示为rd10小鼠p28时来自每个治疗组的单个小鼠的一只眼睛的代表性ERG波形[2.2Log（cd.s/m2）]。图48B所示为每个治疗组ERG a波和b波的平均振幅（蓝色表示rd10-sEVs与rd10-PBS的统计学差异，红色表示rd10-sIVs与rd10-PBS的统计学差异，#表示rd10-sEVs与rd10-sIVs之间的统计学差异。p＜0.05、p＜0.001和p＜0.0001。##p＜0.01、###p＜0.001和####p＜0.0001）。Figure 48 shows that MSC-sIVs rescued the electrophysiological function of the retina of rd10 mice. Figure 48A shows a representative ERG waveform from one eye of a single mouse in each treatment group at p28 in rd10 mice [2.2Log (cd.s/m2)]. Figure 48B shows the average amplitude of the ERG a-wave and b-wave in each treatment group (blue Indicates the statistical difference between rd10-sEVs and rd10-PBS, red indicates the statistical difference between rd10-sIVs and rd10-PBS,# indicates the statistical difference between rd10-sEVs and rd10-sIVs. p＜0.05, p<0.001 and p＜0.0001.## p＜0.01,### p＜0.001 and#### p＜0.0001).
图49所示为MSC-sIVs减少rd10小鼠的视网膜凋亡。图49A所示为各组具有代表性的形态学图像；绿色信号是TUNEL，蓝色信号是DAPI染色细胞核。比例尺：20μm。图49B所示为从整个视网膜中计数ONL中的TUNEL阳性细胞核（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。FIG49 shows that MSC-sIVs reduce retinal apoptosis in rd10 mice. FIG49A shows representative morphological images of each group; green signal is TUNEL, blue signal is DAPI-stained cell nuclei. Scale bar: 20 μm. FIG49B shows the number of TUNEL-positive cell nuclei in ONL counted from the entire retina ( p＜0.05, p＜0.01, p<0.001 and p＜0.0001 indicates significant difference among the groups).
图50所示为MSC-sIVs挽救rd10小鼠的视网膜光感受器细胞丢失。图50A所示为用免疫荧光法测定小鼠视网膜中视紫红质蛋白的表达。细胞核用DAPI（蓝色）染色。比例尺：20μm。图50B所示为荧光定量分析视网膜中视紫红质蛋白的相对表达（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。FIG50 shows that MSC-sIVs rescued the loss of retinal photoreceptor cells in rd10 mice. FIG50A shows the expression of rhodopsin protein in the mouse retina measured by immunofluorescence. The nuclei were stained with DAPI (blue). Scale bar: 20 μm. FIG50B shows the relative expression of rhodopsin protein in the retina by fluorescence quantitative analysis ( p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).
图51所示为MSC-sIVs挽救rd10小鼠的视网膜突触丢失。图51A所示为用免疫荧光法测定小鼠视网膜中PSD95蛋白的表达。细胞核用DAPI（蓝色）染色。比例尺：20μm。图51B所示为荧光定量分析视网膜中PSD95蛋白的相对表达（p＜0.05、p＜0.01和p＜0.0001表示各组间显著性差异）。FIG51 shows that MSC-sIVs rescued retinal synaptic loss in rd10 mice. FIG51A shows the expression of PSD95 protein in the mouse retina measured by immunofluorescence. Cell nuclei were stained with DAPI (blue). Scale bar: 20 μm. FIG51B shows the relative expression of PSD95 protein in the retina by fluorescence quantitative analysis ( p＜0.05, p<0.01 and p<0.0001 indicates significant difference among the groups).
图52所示为MSC-sIVs减少rd10小鼠的Müller细胞活化。图52A所示为免疫荧光法用于测定小鼠视网膜中GFAP的表达。细胞核用DAPI（蓝色）染色。比例尺：20μm。图52B所示为视网膜中GFAP的相对表达（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。FIG52 shows that MSC-sIVs reduce Müller cell activation in rd10 mice. FIG52A shows that immunofluorescence was used to determine the expression of GFAP in the mouse retina. Cell nuclei were stained with DAPI (blue). Scale bar: 20 μm. FIG52B shows the relative expression of GFAP in the retina ( p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).
图53所示为MSC-sIVs减少rd10小鼠视网膜小胶质细胞增殖。图53A所示为Iba1阳性细胞在视网膜中的分布。比例尺：20μm。图53B所示为全视网膜、内层视网膜和外层视网膜Iba1阳性细胞的定量分析（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。FIG53 shows that MSC-sIVs reduce the proliferation of retinal microglia in rd10 mice. FIG53A shows the distribution of Iba1-positive cells in the retina. Scale bar: 20 μm. FIG53B shows the quantitative analysis of Iba1-positive cells in the whole retina, inner retina and outer retina ( p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).
图54所示为免疫荧光染色Iba1显示小胶质细胞在视网膜切片中的分布。比例尺：20μm。Figure 54 shows the distribution of microglia in retinal sections by immunofluorescence staining of Iba1. Scale bar: 20 μm.
图55所示为视网膜中GFAP、PSD95和视紫红质的蛋白质印迹分析。其中，图55A和图55B分别所示为小鼠视网膜中GFAP、PSD95和视紫红质蛋白的蛋白质印迹分析和定量分析（p＜0.05、p＜0.01、p＜0.001和p＜0.0001表示各组间显著性差异）。FIG55 shows the Western blot analysis of GFAP, PSD95 and rhodopsin in the retina. FIG55A and FIG55B show the Western blot analysis and quantitative analysis of GFAP, PSD95 and rhodopsin proteins in the mouse retina, respectively. p＜0.05, p＜0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).
图56所示为MSC-sIVs相较于PBS作用于光损伤小鼠视网膜的差异基因通路富集分析。FIG56 shows the differential gene pathway enrichment analysis of MSC-sIVs compared with PBS on the retina of light-damaged mice.
图57所示为MSC-sIVs相较于PBS作用于rd10小鼠视网膜的差异基因通路富集分析。FIG. 57 shows the differential gene pathway enrichment analysis of MSC-sIVs compared with PBS in the retina of rd10 mice.
图58所示为MSC-sEVs和MSC-sIVs中总蛋白的GSVA富集分析热图。FIG58 shows a heat map of GSVA enrichment analysis of total proteins in MSC-sEVs and MSC-sIVs.
图59所示为MSC-sEVs和MSC-sIVs中总丰度Top50的miRNA表达情况。FIG. 59 shows the expression of the top 50 miRNAs in terms of total abundance in MSC-sEVs and MSC-sIVs.
图60所示为MSC-sEVs和MSC-sIVs中总丰度Top50的差异miRNA靶基因富集分析。Figure 60 shows the differential enrichment analysis of the top 50 total abundance miRNA target genes in MSC-sEVs and MSC-sIVs.
图61所示为蓝光照射小鼠视网膜后在不同时间点的内质网应激标志蛋白表达情况。其中，图61A所示为通过Western Blot检测小鼠视网膜GRP78蛋白表达情况示意图，β-actin作为内参。图61B所示为GRP78统计学结果。（n=6,p<0.05、p<0.01、p<0.001和p<0.0001表示与NoBL-PBS组相比的统计学差异）。Figure 61 shows the expression of endoplasmic reticulum stress marker proteins at different time points after blue light irradiation of mouse retina. Figure 61A shows the schematic diagram of GRP78 protein expression in mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 61B shows the statistical results of GRP78. (n=6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference compared with the NoBL-PBS group).
图62所示为MSC-sIVs抑制蓝光损伤小鼠视网膜内质网应激。其中，图62A所示为通过Western Blot检测小鼠视网膜靶蛋白表达情况示意图，β-actin作为内参。图62B所示为GRP78统计学结果。图62C所示为p-PERK/PERK的统计学结果。图62D所示为p-PERK的统计学结果。图62E所示为PERK的统计学结果。图62F所示为IRE1a的统计学结果。图62G所示为ATF6的统计学结果。图62H所示为p-eIF2a/eIF2a的统计学结果。图62I所示为p-eIF2a的统计学结果。图62J所示为eIF2a的统计学结果。图62K所示为CHOP的统计学结果。（n=4-6,p<0.05、p<0.01、p<0.001和p<0.0001表示与NoBL-PBS组相比的统计学差异，#p<0.05、##p<0.01、###p<0.001和####p<0.0001表示与BL-PBS组相比的统计学差异，&p<0.05、&&p<0.01、&&&p<0.001和&&&&p<0.0001表示同剂量sEVs与sIVs之间的统计学差异）。Figure 62 shows that MSC-sIVs inhibits endoplasmic reticulum stress in the retina of mice damaged by blue light. Figure 62A shows a schematic diagram of the expression of target proteins in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 62B shows the statistical results of GRP78. Figure 62C shows the statistical results of p-PERK/PERK. Figure 62D shows the statistical results of p-PERK. Figure 62E shows the statistical results of PERK. Figure 62F shows the statistical results of IRE1a. Figure 62G shows the statistical results of ATF6. Figure 62H shows the statistical results of p-eIF2a/eIF2a. Figure 62I shows the statistical results of p-eIF2a. Figure 62J shows the statistical results of eIF2a. Figure 62K shows the statistical results of CHOP. (n=4-6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates statistical difference compared with the NoBL-PBS group,# p<0.05,## p<0.01,### p<0.001 and#### p<0.0001 indicate statistical difference compared with the BL-PBS group,& p<0.05,&& p<0.01,&&& p<0.001 and&&&& p<0.0001 indicate statistical difference between sEVs and sIVs with the same dose).
图63所示为MSC-sIVs抑制蓝光损伤小鼠视网膜凋亡信号。其中，图63A所示为通过Western Blot检测小鼠视网膜靶蛋白表达情况示意图，β-actin作为内参。图63B所示为Cleaved-Caspase3/-Caspase3统计学结果。图63C所示为Caspase3的统计学结果。图63D所示为Bax的统计学结果。图63E所示为Bcl-2的统计学结果。（n=4-6,p<0.05、p<0.01、p<0.001和p<0.0001表示与NoBL-PBS组相比的统计学差异，#p<0.05、##p<0.01、###p<0.001和####p<0.0001表示与BL-PBS组相比的统计学差异，&p<0.05、&&p<0.01、&&&p<0.001和&&&&p<0.0001表示同剂量sEVs与sIVs之间的统计学差异）。Figure 63 shows that MSC-sIVs inhibits apoptosis signals in the retina of mice damaged by blue light. Figure 63A shows a schematic diagram of the expression of target proteins in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 63B shows the statistical results of Cleaved-Caspase3/-Caspase3. Figure 63C shows the statistical results of Caspase3. Figure 63D shows the statistical results of Bax. Figure 63E shows the statistical results of Bcl-2. (n=4-6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates statistical difference compared with the NoBL-PBS group,# p<0.05,## p<0.01,### p<0.001 and#### p<0.0001 indicate statistical difference compared with the BL-PBS group,& p<0.05,&& p<0.01,&&& p<0.001 and&&&& p<0.0001 indicate statistical difference between sEVs and sIVs with the same dose).
图64所示为rd10小鼠视网膜在不同时间点的内质网应激标志蛋白表达情况。其中，图64A所示为通过Western Blot检测小鼠视网膜GRP78蛋白表达情况示意图，β-actin作为内参。图64B所示为GRP78统计学结果。（n=4,p<0.05、p<0.01、p<0.001和p<0.0001表示两组相比的统计学差异）。Figure 64 shows the expression of endoplasmic reticulum stress marker proteins in the retina of rd10 mice at different time points. Figure 64A shows the schematic diagram of the expression of GRP78 protein in the mouse retina detected by Western Blot, with β-actin as the internal reference. Figure 64B shows the statistical results of GRP78. (n=4, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference between the two groups).
图65所示为MSC-sIVs抑制rd10小鼠p21视网膜内质网应激。其中，图65A所示为通过Western Blot检测小鼠视网膜靶蛋白表达情况示意图，β-actin作为内参。图65B所示为GRP78统计学结果。图65C所示为p-PERK/PERK的统计学结果。图65D所示为p-PERK的统计学结果。图65E所示为PERK的统计学结果。图65F所示为IRE1a的统计学结果。图65G所示为ATF6的统计学结果。图65H所示为p-eIF2a/eIF2a的统计学结果。图65I所示为p-eIF2a的统计学结果。图65J所示为eIF2a的统计学结果。图65K所示为CHOP的统计学结果。（n=4-6,p<0.05、p<0.01、p<0.001和p<0.0001表示两组相比的统计学差异）。Figure 65 shows that MSC-sIVs inhibits endoplasmic reticulum stress in the retina of p21 rd10 mice. Figure 65A shows a schematic diagram of the expression of target proteins in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 65B shows the statistical results of GRP78. Figure 65C shows the statistical results of p-PERK/PERK. Figure 65D shows the statistical results of p-PERK. Figure 65E shows the statistical results of PERK. Figure 65F shows the statistical results of IRE1a. Figure 65G shows the statistical results of ATF6. Figure 65H shows the statistical results of p-eIF2a/eIF2a. Figure 65I shows the statistical results of p-eIF2a. Figure 65J shows the statistical results of eIF2a. Figure 65K shows the statistical results of CHOP. (n=4-6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference between the two groups).
图66所示为MSC-sIVs抑制rd10小鼠p21视网膜凋亡信号。其中，图66A所示为通过Western Blot检测小鼠视网膜靶蛋白表达情况示意图，β-actin作为内参。图66B所示为Cleaved-Caspase3/-Caspase3统计学结果。图66C所示为Caspase3的统计学结果。图66D所示为Bax的统计学结果。图66E所示为Bcl-2的统计学结果。（n=3-6，p<0.05、p<0.01、p<0.001和p<0.0001表示两组相比的统计学差异）。Figure 66 shows that MSC-sIVs inhibited the apoptosis signal of retina p21 in rd10 mice. Figure 66A shows the expression of target proteins in mouse retina detected by Western Blot, with β-actin as internal reference. Figure 66B shows the statistical results of Cleaved-Caspase3/-Caspase3. Figure 66C shows the statistical results of Caspase3. Figure 66D shows the statistical results of Bax. Figure 66E shows the statistical results of Bcl-2. (n=3-6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference between the two groups).
图67所示为MSC-sIVs抑制rd10小鼠p28视网膜内质网应激。其中，图67A所示为通过Western Blot检测小鼠视网膜靶蛋白表达情况示意图，β-actin作为内参。图67B所示为GRP78统计学结果。图67C所示为p-PERK/PERK的统计学结果。图67D所示为p-PERK的统计学结果。图67E所示为PERK的统计学结果。图67F所示为IRE1a的统计学结果。图67G所示为ATF6的统计学结果。图67H所示为p-eIF2a/eIF2a的统计学结果。图67I所示为p-eIF2a的统计学结果。图67J所示为eIF2a的统计学结果。图67K所示为CHOP的统计学结果。（n=4-6,p<0.05、p<0.01、p<0.001和p<0.0001表示两组相比的统计学差异）。Figure 67 shows that MSC-sIVs inhibits endoplasmic reticulum stress in the p28 retina of rd10 mice. Figure 67A shows a schematic diagram of the expression of target proteins in the mouse retina detected by Western Blot, with β-actin as an internal reference. Figure 67B shows the statistical results of GRP78. Figure 67C shows the statistical results of p-PERK/PERK. Figure 67D shows the statistical results of p-PERK. Figure 67E shows the statistical results of PERK. Figure 67F shows the statistical results of IRE1a. Figure 67G shows the statistical results of ATF6. Figure 67H shows the statistical results of p-eIF2a/eIF2a. Figure 67I shows the statistical results of p-eIF2a. Figure 67J shows the statistical results of eIF2a. Figure 67K shows the statistical results of CHOP. (n=4-6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference between the two groups).
图68所示为MSC-sIVs抑制rd10小鼠p28视网膜凋亡信号。其中，图68A所示为通过Western Blot检测小鼠视网膜靶蛋白表达情况示意图，β-actin作为内参。图68B所示为Cleaved-Caspase3/-Caspase3统计学结果。图68C所示为Caspase3的统计学结果。图68D所示为Bax的统计学结果。图68E所示为Bcl-2的统计学结果。（n=3-6，p<0.05、p<0.01、p<0.001和p<0.0001表示两组相比的统计学差异）。Figure 68 shows that MSC-sIVs inhibited the apoptosis signal of p28 retina in rd10 mice. Figure 68A shows the expression of target proteins in mouse retina detected by Western Blot, with β-actin as the internal reference. Figure 68B shows the statistical results of Cleaved-Caspase3/-Caspase3. Figure 68C shows the statistical results of Caspase3. Figure 68D shows the statistical results of Bax. Figure 68E shows the statistical results of Bcl-2. (n=3-6, p<0.05, p<0.01, p<0.001 and p<0.0001 indicates a statistically significant difference between the two groups).
图69所示为不同浓度梯度sIVs和sEVs作用24小时后对HCEC增殖的作用。对照组为PBS治疗组。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。#表示sIVs与对照组的差异，#p<0.05、##p<0.01、###p<0.001、####p<0.0001。Figure 69 shows the effect of different concentration gradients of sIVs and sEVs on HCEC proliferation after 24 hours. The control group was a PBS treatment group. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001. # indicates the difference between sIVs and control group,# p<0.05,## p<0.01,### p<0.001,#### p<0.0001.
图70所示为不同浓度梯度sIVs和sEVs作用24小时后对HCEC增殖的作用折线图。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。FIG70 is a line graph showing the effect of different concentration gradients of sIVs and sEVs on HCEC proliferation after 24 hours. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001.
图71所示为不同浓度梯度sIVs和sEVs作用48小时后对HCEC增殖的作用。对照组为PBS治疗组。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。#表示sIVs或sEVs与对照组的差异，#p<0.05、##p<0.01、###p<0.001、####p<0.0001。Figure 71 shows the effect of different concentration gradients of sIVs and sEVs on HCEC proliferation after 48 hours. The control group was a PBS treatment group. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001. # indicates the difference between sIVs or sEVs and the control group,# p<0.05,## p<0.01,### p<0.001,#### p<0.0001.
图72所示为不同浓度梯度sIVs和sEVs作用48小时后对HCEC增殖作用的折线图。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。FIG72 is a line graph showing the effect of different concentration gradients of sIVs and sEVs on HCEC proliferation after 48 hours. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001.
图73所示为20μg/ml的sIVs和sEVs对HCEC的增殖作用结果。对照组为PBS治疗组。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。#表示sIVs或sEVs与对照组的差异，#p<0.05、##p<0.01、###p<0.001、####p<0.0001。Figure 73 shows the results of the proliferation effect of 20 μg/ml sIVs and sEVs on HCEC. The control group was a PBS treatment group. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001. # indicates the difference between sIVs or sEVs and the control group,# p<0.05,## p<0.01,### p<0.001,#### p<0.0001.
图74所示为40μg/ml的sIVs和sEVs对HCEC的增殖作用图。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。#表示sIVs或sEVs与对照组的差异，#p<0.05、##p<0.01、###p<0.001、####P ≤0.0001。FIG. 74 is a graph showing the proliferation effect of 40 μg/ml sIVs and sEVs on HCEC. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001. # indicates the difference between sIVs or sEVs and the control group,# p<0.05,## p<0.01,### p<0.001,#### P ≤0.0001.
图75所示为不同浓度梯度sIVs和sEVs作用24小时后对HCEC迁移趋化作用的代表性结果图。对照组为PBS治疗组。紫色示结晶紫染色的细胞，细胞越多表示细胞迁移性越强。Figure 75 shows the representative results of the chemotactic effect of sIVs and sEVs at different concentration gradients on HCEC migration after 24 hours. The control group was the PBS treatment group. Purple indicates crystal violet-stained cells, and the more cells, the stronger the cell migration.
图76所示为不同浓度梯度sIVs和sEVs作用24小时后对HCEC迁移趋化作用统计结果。对照组为PBS治疗组。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。#表示sIVs或sEVs与对照组的差异，#p<0.05、##p<0.01、###p<0.001、####p<0.0001。Figure 76 shows the statistical results of the chemotactic effect of sIVs and sEVs at different concentration gradients on HCEC migration after 24 hours. The control group was the PBS treatment group. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001. # indicates the difference between sIVs or sEVs and the control group,# p<0.05,## p<0.01,### p<0.001,#### p<0.0001.
图77所示为细胞划痕代表性结果图，显示PBS(对照组)、20μg/ml sEVs和20μg/mlsIVs对HCEC迁移的作用。对照组为PBS治疗组。Figure 77 shows representative results of cell scratches, showing the effects of PBS (control group), 20 μg/ml sEVs and 20 μg/ml sIVs on HCEC migration. The control group was the PBS treatment group.
图78所示为伤口闭合比率统计结果，显示PBS(对照组)、20μg/ml sEVs和20μg/mlsIVs作用于HCEC迁移的作用。对照组为PBS治疗组。表示为sIVs与sEVs的统计学差异，p<0.05、p<0.01、p<0.001、p<0.0001。#表示sIVs或sEVs与对照组的差异，#p<0.05、##p<0.01、###p<0.001、####p<0.0001。Figure 78 shows the statistical results of wound closure ratio, showing the effects of PBS (control group), 20 μg/ml sEVs and 20 μg/ml sIVs on HCEC migration. The control group was the PBS treatment group. Expressed as the statistical difference between sIVs and sEVs, p<0.05, p<0.01, p<0.001, p<0.0001. # indicates the difference between sIVs or sEVs and the control group,# p<0.05,## p<0.01,### p<0.001,#### p<0.0001.
图79所示为DiD染色的sIVs和sEVs对小鼠角膜创伤模型作用4小时后冰冻切片，体现缺损的角膜上皮对MSC-sEVs和MSC-sIVs的亲和力。Figure 79 shows the frozen sections of the mouse corneal trauma model 4 hours after the DiD-stained sIVs and sEVs were applied, reflecting the affinity of the defective corneal epithelium to MSC-sEVs and MSC-sIVs.
图80所示为0.1μg/μl的sIVs或sEVs及PBS组对小鼠角膜荧光素钠染色图。对照组为PBS治疗组。Figure 80 shows the corneal fluorescein sodium staining of mice in the 0.1 μg/μl sIVs or sEVs and PBS groups. The control group is the PBS treatment group.
图81所示为0.1μg/μl的sIVs或sEVs及PBS组对小鼠角膜上皮缺损比率，越小代表愈合程度越高。p<0.05、p<0.01、p<0.001。对照组为PBS治疗组。Figure 81 shows the ratio of corneal epithelial defects in mice in the 0.1 μg/μl sIVs or sEVs and PBS groups. The smaller the ratio, the higher the degree of healing. p<0.05, p<0.01, p<0.001. The control group was the PBS treatment group.
图82所示为角膜敏感性阈值统计。数值越大表明角膜恢复后敏感度越高。p<0.05、p<0.01、p<0.001。对照组为PBS治疗组。The statistics of corneal sensitivity threshold are shown in Figure 82. The larger the value, the higher the sensitivity of the cornea after recovery. p<0.05, p<0.01, p<0.001. The control group was the PBS treatment group.
图83所示为裂隙灯下的角膜新生血管生长情况。碱烧伤组血管增粗，出现大量新生血管。对照组为PBS治疗组。Figure 83 shows the growth of corneal neovascularization under slit lamp. The blood vessels in the alkali burn group were thickened and a large number of new blood vessels appeared. The control group was the PBS treatment group.
图84所示为角膜铺片，红色示CD31标记的血管。对照组为PBS治疗组。Figure 84 shows corneal flat mounts, with blood vessels marked by CD31 in red. The control group was the PBS treatment group.
图85所示为角膜新生血管统计学结果，对照组为PBS治疗组。p<0.05、p<0.01、p<0.001、p<0.0001。FIG. 85 shows the statistical results of corneal neovascularization, and the control group is the PBS treatment group. p<0.05, p<0.01, p<0.001, p<0.0001.
具体实施方式DETAILED DESCRIPTION
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.
本发明所述疾病名称参照现有技术，例如国际疾病分类（ICD）-10、11中所述。The names of diseases described in the present invention refer to the prior art, such as those described in the International Classification of Diseases (ICD)-10 and 11.
术语“角膜”是指在眼睛的中间区域的眼球表面上的透明膜，其具有能够保护眼睛免受外部影响并允许光穿过并折射由此允许产生视觉敏锐度的结构。The term "cornea" refers to a transparent membrane on the surface of the eyeball in the middle region of the eye, which has a structure capable of protecting the eye from external influences and allowing light to pass through and refract, thereby allowing visual acuity to occur.
术语“角膜疾病”是指发生在角膜的所有疾病，特别是引起角膜损伤由此导致透明视觉损伤的疾病。角膜疾病可归因于某些内源疾病，如疱疹性角膜炎、细菌性角膜溃疡、神经麻痹性角膜炎、糖尿病性角膜病变、Sjogren综合症，Stevens-Johnson综合症、干性角结膜炎(干眼病)等，也可能归因于外源疾病，如手术后、药物诱发、创伤和贴眼睛透镜佩戴者的疾病，或由物理或化学损伤引起的疾病。The term "corneal disease" refers to all diseases occurring in the cornea, particularly those causing damage to the cornea, thereby causing damage to clear vision. Corneal diseases may be attributed to certain endogenous diseases, such as herpetic keratitis, bacterial corneal ulcers, neuroparetic keratitis, diabetic keratopathy, Sjogren's syndrome, Stevens-Johnson syndrome, keratoconjunctivitis sicca (dry eye), etc., or to exogenous diseases, such as post-operative, drug-induced, traumatic and conjunctivitis in eye lens wearers, or diseases caused by physical or chemical damage.
术语“视网膜疾病”是指发生在视网膜的所有疾病，特别是视网膜损伤和变性及其相关疾病。The term "retinal disease" refers to all diseases occurring in the retina, particularly retinal damage and degeneration and related diseases.
术语“患者”或“受试者”等等在本文中可交换使用，是指根据本文所述的方法治疗的任何动物或其细胞，不论是体外或原位。具体地，前述动物包括哺乳动物，例如，大鼠、小鼠、豚鼠、兔、犬、猴子、人类，特别是人类。The term "patient" or "subject" and the like are used interchangeably herein and refer to any animal or cell thereof treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, e.g., rats, mice, guinea pigs, rabbits, dogs, monkeys, humans, particularly humans.
术语“治疗”是指在疾病发作之后预防、治愈、逆转、减弱、减轻、最小化、抑制、制止和/或停止疾病的一种或多种临床症状。The term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and/or halting one or more clinical symptoms of a disease after onset of the disease.
术语“预防”指在疾病发作之前，通过治疗以避免、最小化或令疾病难于发作或发展。The term "prevent" refers to avoiding, minimizing or making the onset or development of a disease difficult by treating it before it occurs.
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
实施例1：间充质干细胞（MSC）的提取及培养Example 1: Extraction and culture of mesenchymal stem cells (MSC)
人脐带由北京贝来生物科技有限公司提供，从剖宫产后没有并发症的正常妊娠中获得的脐带立即放入含有青霉素（100U/mL）和链霉素（100mg/mL）的盐水中，然后在4小时内运送到实验室。在去除残余血液和血管后，将获得的脐带切成1-3mm的块，并用0.1%的Ⅱ型胶原酶在37℃下消化1小时。然后通过100目筛网过滤悬浮液以去除未消化的组织。将来自过滤的上清液离心并用PBS洗涤三次。将细胞沉淀重悬于Dulbecco改良的Eagle培养基/营养混合物F12完全培养基中。培养基含有10%胎牛血清（FBS）、100U/ml青霉素和100mg/ml链霉素。将细胞接种在T175培养瓶中，并在37℃的5% CO2培养箱中培养。培养基每3天更换一次。当细胞融合达到80%时，以1:2的继代比例进行传代，并使用P3至P5的细胞进行实验。Human umbilical cords were provided by Beijing Beilai Biotechnology Co., Ltd. Umbilical cords obtained from normal pregnancies without complications after cesarean section were immediately placed in saline containing penicillin (100 U/mL) and streptomycin (100 mg/mL) and then transported to the laboratory within 4 hours. After removing residual blood and blood vessels, the obtained umbilical cords were cut into 1-3 mm pieces and digested with 0.1% type II collagenase for 1 hour at 37°C. The suspension was then filtered through a 100-mesh screen to remove undigested tissue. The supernatant from the filtration was centrifuged and washed three times with PBS. The cell pellet was resuspended in Dulbecco's modified Eagle medium/nutrient mixture F12 complete medium. The culture medium contained 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The cells were seeded in T175 culture flasks and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed every 3 days. When cell confluence reached 80%, cells were passaged at a 1:2 subculture ratio, and cells from P3 to P5 were used for experiments.
实施例2：细胞来源的纳米囊泡的制备Example 2: Preparation of cell-derived nanovesicles
细胞内纳米囊泡的生产方法流程如图1所示。The process flow of the method for producing intracellular nanovesicles is shown in FIG1 .
1、细胞消化与计数1. Cell digestion and counting
细胞生长到90%融合，吸净细胞上清，加入PBS清洗细胞2次，加入胰酶消化细胞，消化后使用PBS中和并清洗细胞3次。随后进行细胞计数，使用PBS将细胞数量调整至1×106个/mL。When the cells grew to 90% confluence, the cell supernatant was aspirated, the cells were washed twice with PBS, and the cells were digested with trypsin. After digestion, the cells were neutralized and washed three times with PBS. The cells were then counted and the cell number was adjusted to 1×106 /mL with PBS.
2、超声处理细胞2. Ultrasonic treatment of cells
取1×106个/mL密度的细胞悬液2 mL，加至50 mL离心管的底部，将超声探头放入液面中央，超声振幅参数为20%，时间参数为15s，on 2s，off 2s，离心管置于冰上。随后将该液体转移至2mL离心管进行离心。Take 2 mL of cell suspension with a density of 1×106 cells/mL and add it to the bottom of a 50 mL centrifuge tube. Place the ultrasonic probe in the center of the liquid surface. The ultrasonic amplitude parameter is 20%, the time parameter is 15s, on 2s, off 2s, and the centrifuge tube is placed on ice. Then transfer the liquid to a 2 mL centrifuge tube for centrifugation.
3、小细胞内纳米囊泡的收集3. Collection of Nanovesicles in Small Cells
离心参数是2000g×10min，20000g×30min。随后收集上清转移至超速离心管，离心参数是150000g×70min，以上操作均在冰上进行。获得的沉淀使用PBS重悬，为小细胞内纳米囊泡（small Intracellular Nanovesicles, sIVs）。The centrifugation parameters were 2000g×10min, 20000g×30min. The supernatant was then collected and transferred to an ultracentrifuge tube, and the centrifugation parameters were 150000g×70min. All the above operations were performed on ice. The obtained precipitate was resuspended in PBS and was small intracellular nanovesicles (sIVs).
4、小细胞外囊泡的收集4. Collection of small extracellular vesicles
FBS是体外培养细胞的必需条件，但是其内含有大量牛来源的细胞外囊泡，也会存在于含FBS的细胞完全培养基中。为了去除牛细胞外囊泡，将FBS在4℃下以110000×g离心过夜（约12小时）。当细胞融合达到60%时，将细胞在含有10%去除外泌体的FBS的完全培养基中培养48小时。然后，收集上清液，并通过在4℃下超速离心分离细胞外囊泡。具体步骤包括300g×10min、2000g×10min、10000g×30min和110000g×70min两次。获得的沉淀使用PBS重悬，为以外泌体为主的小细胞外囊泡（small Extracellular Vesicles, sEVs）。FBS is a necessary condition for in vitro cell culture, but it contains a large number of bovine extracellular vesicles, which will also be present in the complete cell culture medium containing FBS. In order to remove bovine extracellular vesicles, FBS was centrifuged at 110,000×g at 4°C overnight (about 12 hours). When the cell fusion reached 60%, the cells were cultured in complete culture medium containing 10% FBS with exosomes removed for 48 hours. Then, the supernatant was collected and the extracellular vesicles were separated by ultracentrifugation at 4°C. The specific steps included 300g×10min, 2000g×10min, 10000g×30min and 110000g×70min twice. The obtained precipitate was resuspended in PBS, which was small extracellular vesicles (sEVs) mainly composed of exosomes.
实施例3：对细胞内纳米囊泡的分离参数的优化Example 3: Optimization of isolation parameters for intracellular nanovesicles
参照实施例2的步骤，对细胞内纳米囊泡的分离参数的优化过程如图2所示。Referring to the steps of Example 2, the optimization process of the separation parameters of intracellular nanovesicles is shown in FIG2 .
1、纳米粒度分析仪检测sIVs的产量差异1. Nanoparticle size analyzer detects the difference in the yield of sIVs
取PBS重悬的sIVs，用PBS稀释至1ml，使用纳米颗粒跟踪分析软件（NanoparticleTracking Analysis，NTA）NTA 3.3 Dev Build 3.3.104进行检测，设置温度为25℃，激光设置为Blue488，流速设置为50，模式为自动检测，进样三次，分析三次，取峰值平均值为Mode粒径结果。相机模式为sCMOS。激光类型为Blue488，粘滞度为0.9cP。Take the PBS-resuspended sIVs, dilute them to 1 ml with PBS, and use the Nanoparticle Tracking Analysis (NTA) software NTA 3.3 Dev Build 3.3.104 for detection. Set the temperature to 25°C, the laser to Blue488, the flow rate to 50, the mode to automatic detection, inject three times, analyze three times, and take the peak value average as the Mode particle size result. The camera mode is sCMOS. The laser type is Blue488, and the viscosity is 0.9 cP.
2、透射电镜观察不同细胞sIVs的形态2. Observation of the morphology of sIVs in different cells by transmission electron microscopy
取离心后的sIVs，重悬于200μL PBS液中混匀，取10μL的sIVs溶液和4%PFA按体积比1：1混合，滴在干净的塑料薄膜上形成液滴，然后将电镜碳网的正面扣在液滴上，放置20min，10μL磷钨酸负染90s，烤干碳网，使用HitacW-7500透射电子显微镜进行观察。Take the centrifuged sIVs, resuspend them in 200μL PBS solution and mix them evenly. Take 10μL sIVs solution and mix it with 4% PFA in a volume ratio of 1:1, drop it on a clean plastic film to form droplets, then put the front of the electron microscope carbon grid on the droplets and leave it for 20 minutes. Negatively stain with 10μL phosphotungstic acid for 90s, bake the carbon grid dry, and observe it using a HitacW-7500 transmission electron microscope.
图2A和图2B分别显示在20%的超声振幅下，选择5s、10s、15s、20s、25s、30s和60s的作用时间，按照本实施步骤所得sIVs的蛋白质产量和囊泡产量。结果显示作用时间低于10秒或超过20秒，所得囊泡的数量和蛋白质产量骤降。图2C和图2D分别显示在15s的超声作用时间下，选择20%、25%、30%、35%和40%的振幅，按照本实施步骤所得sIVs的蛋白质产量和囊泡产量。结果显示在15s的超声时间下，超声振幅高于25%，囊泡产量骤降。图2E示在15s的超声时间下，选择20%、25%、30%、35%和40%的超声振幅，按照本实施步骤所得sIVs的透射电镜图。图2F示在20%的超声振幅下，选择5s、10s、15s、20s、25s、30s和60s的超声时间，按照本实施步骤所得sIVs的透射电镜图。Figures 2A and 2B show the protein yield and vesicle yield of sIVs obtained according to the implementation steps at an ultrasonic amplitude of 20%, with an action time of 5s, 10s, 15s, 20s, 25s, 30s and 60s, respectively. The results show that when the action time is less than 10 seconds or more than 20 seconds, the number of vesicles and protein yield obtained drop sharply. Figures 2C and 2D show the protein yield and vesicle yield of sIVs obtained according to the implementation steps at an ultrasonic action time of 15s, with an amplitude of 20%, 25%, 30%, 35% and 40%, respectively. The results show that when the ultrasonic amplitude is higher than 25% at an ultrasonic time of 15s, the vesicle yield drops sharply. Figure 2E shows a transmission electron micrograph of sIVs obtained according to the implementation steps at an ultrasonic time of 15s, with an ultrasonic amplitude of 20%, 25%, 30%, 35% and 40%. FIG2F shows transmission electron micrographs of sIVs obtained according to the present implementation steps at an ultrasonic amplitude of 20% and ultrasonic times of 5 s, 10 s, 15 s, 20 s, 25 s, 30 s and 60 s.
本优化过程表明，在20%的超声振幅下，作用时间超过低于10秒或20秒，囊泡产量骤降。在15s的超声时间下，超声振幅高于25%，囊泡产量骤降。20%的振幅以及15s的超声时间是最佳的收集细胞内纳米囊泡的参数。This optimization process shows that at an ultrasonic amplitude of 20%, the vesicle yield drops sharply when the action time exceeds 10 seconds or 20 seconds. At an ultrasonic time of 15 seconds, the vesicle yield drops sharply when the ultrasonic amplitude is higher than 25%. 20% amplitude and 15 seconds ultrasonic time are the best parameters for collecting intracellular nanovesicles.
实施例4：sIVs具有独特的物理特征和较高的热稳定性Example 4: sIVs have unique physical characteristics and high thermal stability
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表1实验试剂Table 1 Experimental reagents
1.2、实验仪器1.2 Experimental instruments
表2实验仪器Table 2 Experimental instruments
2、实验方法2. Experimental methods
2.1透射电镜观察sIVs和sIVs的形态2.1 Transmission electron microscopy observation of the morphology of sIVs and sIVs
取离心后的sEVs和sIVs（实施例2制备），重悬于200μL PBS液中混匀，取10μL的sEVs和sIVs溶液和4% PFA按体积比1：1混合，滴在干净的塑料薄膜上形成液滴，然后将碳网的正面扣在液滴上，放置20min，10μL磷钨酸负染90s，烤干碳网，使用HitacW-7500透射电子显微镜进行观察。Take the centrifuged sEVs and sIVs (prepared in Example 2), resuspend them in 200 μL PBS solution and mix them evenly. Take 10 μL of sEVs and sIVs solution and mix them with 4% PFA in a volume ratio of 1:1, drop them on a clean plastic film to form droplets, then put the front side of the carbon grid on the droplets and leave it for 20 minutes. Negatively stain with 10 μL phosphotungstic acid for 90 seconds, bake the carbon grid dry, and observe it using a HitacW-7500 transmission electron microscope.
2.2纳米粒度分析检测sEVs和sIVs的粒子直径2.2 Nanoparticle size analysis to detect the particle diameter of sEVs and sIVs
取PBS重悬的sEVs和sIVs，用PBS稀释至1ml，使用纳米颗粒跟踪分析软件（Nanoparticle Tracking Analysis，NTA）3.3 Dev Build 3.3.104进行检测，设置温度为25℃，激光设置为Blue 488，流速设置为50，模式设为自动检测，进样三次，分析三次，取峰值平均值为Mode的粒径结果。相机模式设为sCMOS。激光类型设为Blue488，粘滞度设为0.9cP。Take the sEVs and sIVs resuspended in PBS, dilute them to 1 ml with PBS, and use Nanoparticle Tracking Analysis (NTA) 3.3 Dev Build 3.3.104 for detection. Set the temperature to 25°C, the laser to Blue 488, the flow rate to 50, the mode to automatic detection, inject three times, analyze three times, and take the peak value average as the particle size result of Mode. The camera mode is set to sCMOS. The laser type is set to Blue488, and the viscosity is set to 0.9 cP.
2.3考马斯亮蓝染色分析sEVs和sIVs的蛋白构成2.3 Analysis of protein composition of sEVs and sIVs by Coomassie Brilliant Blue staining
经BCA定量后，取等量的蛋白样品，加入PBS定容到20 μl，加入5 μl蛋白上样缓冲液（5×），95℃加热，5分钟。蛋白电泳条件为100V，90min。电泳后加入考马斯亮蓝超快染色液，室温孵育2小时，使用纯水清洗至水变澄清。对胶进行拍摄。After BCA quantification, take an equal amount of protein sample, add PBS to 20 μl, add 5 μl protein loading buffer (5×), and heat at 95°C for 5 minutes. The protein electrophoresis conditions are 100V, 90min. After electrophoresis, add Coomassie Brilliant Blue Ultrafast Staining Solution, incubate at room temperature for 2 hours, and wash with pure water until the water becomes clear. Photograph the gel.
2.4 Western Blot分析sIVs和sEVs的外泌体标志物蛋白构成2.4 Western Blot analysis of exosomal marker protein composition of sIVs and sEVs
经BCA试剂盒对蛋白进行检测后，取等量的各组蛋白样品，加入PBS定容至等体积，加入蛋白上样缓冲液（5×），95℃加热五分钟。按照SDS-PAGE试剂盒说明配置胶，并插入电泳梳。静置并且在室温放置25分钟等待凝固。将制备好的SDS凝胶，放置在预先准备的电泳槽中。除去电泳梳，将变性后的蛋白注入SDS-PAGE的上样槽中。两侧各加入1-4 μl的marker。调整电压为60V，待上层胶跑完后，将电压变成100V，电泳至最下方溴酚蓝指示线距离玻璃板底部1-2cm处停止电泳。将激活后的PVDF膜放置在SDS-PAGE表面上，然后在胶和膜的两侧分别放置滤纸和海绵垫。使用柱子轻轻滚动以去除体系中的气泡，并夹紧电转夹。设置黑色电极为SDS-PAGE，红色电极为PVDF膜，并在恒压下转膜，同时在电转槽中放置冰袋降温。电转结束后，将PVDF膜放入含有5%脱脂牛奶或1% BSA的TBST溶液中，于室温下封闭2小时。封闭完成后，加入一抗，4℃冰箱摇床上过夜孵育。次日，去除一抗，加入TBST溶液洗涤PVDF膜。接着，加入二抗，室温摇床上孵育2小时。孵育结束后，再次去除二抗，加入TBST溶液洗涤PVDF膜3次，每次10分钟。最后，使用ECL超敏发光液对PVDF膜进行显影处理。After protein detection by BCA kit, take equal amounts of protein samples from each group, add PBS to equal volume, add protein loading buffer (5×), and heat at 95℃ for five minutes. Prepare gel according to the instructions of SDS-PAGE kit and insert electrophoresis comb. Let it stand and wait for solidification at room temperature for 25 minutes. Place the prepared SDS gel in the pre-prepared electrophoresis tank. Remove the electrophoresis comb and inject the denatured protein into the SDS-PAGE loading tank. Add 1-4 μl of marker on each side. Adjust the voltage to 60V. After the upper gel runs, change the voltage to 100V and stop electrophoresis until the lowest bromophenol blue indicator line is 1-2cm away from the bottom of the glass plate. Place the activated PVDF membrane on the SDS-PAGE surface, and then place filter paper and sponge pads on both sides of the gel and membrane. Use the column to roll gently to remove bubbles in the system and clamp the electrotransfer clamp. Set the black electrode as SDS-PAGE and the red electrode as PVDF membrane, and transfer the membrane under constant pressure, while placing ice bags in the electrotransfer tank to cool down. After electrotransfer, place the PVDF membrane in a TBST solution containing 5% skim milk or 1% BSA and block it at room temperature for 2 hours. After blocking, add the primary antibody and incubate overnight on a shaker in a 4°C refrigerator. The next day, remove the primary antibody and add TBST solution to wash the PVDF membrane. Then, add the secondary antibody and incubate on a shaker at room temperature for 2 hours. After incubation, remove the secondary antibody again and add TBST solution to wash the PVDF membrane 3 times, 10 minutes each time. Finally, use ECL supersensitive luminescent liquid to develop the PVDF membrane.
3、统计学处理3. Statistical processing
实验数据以均数±标准差（）表示。实验数据均进行正态检验。使用SPSS22.0分析所有定量数据。方差分析采取One-way ANOVA进行，使用最小显着性差异（LSD）分析进行事后检验。对于非正态分布数据和方差不均的数据，使用了非参数检验，P值<0.05为统计学显著差异。The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. All quantitative data were analyzed using SPSS22.0. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, nonparametric tests were used, andP values < 0.05 were considered statistically significant.
4、实验结果4. Experimental results
4.1透射电镜显示sEVs与sIVs的形态4.1 Transmission electron microscopy reveals the morphology of sEVs and sIVs
富集MSCs细胞的sIVs，并使用透射电镜检测。结果如图3所示，MSCs细胞的sEVs呈圆形或马蹄形，直径在100-200nm，sIVs数量较多，形态呈圆形，直径小于100nm。电镜结果显示sIVs的直径明显小于sEVs。MSCs cells were enriched for sIVs and detected using transmission electron microscopy. As shown in Figure 3, MSCs cells' sEVs were round or horseshoe-shaped with a diameter of 100-200 nm, and sIVs were numerous, round in shape, and less than 100 nm in diameter. Electron microscopy results showed that the diameter of sIVs was significantly smaller than that of sEVs.
4.2纳米颗粒跟踪分析显示sEVs和sIVs粒径分布4.2 Nanoparticle tracking analysis reveals the size distribution of sEVs and sIVs
纳米粒度检测结果如图4所示，MSCs细胞的sEVs粒径分布范围广，粒径较大，而sIVs的粒径分布范围窄，粒径较小。The results of nanoparticle size detection are shown in Figure 4 . The particle size distribution range of sEVs from MSCs cells is wide and the particle size is large, while the particle size distribution range of sIVs is narrow and the particle size is small.
经过统计分析，结果如图5所示，MSC的sEVs平均粒径为123.1±4.453nm，sIVs平均粒径为75.28±9.067nm；sIVs粒径小于其sEVs。After statistical analysis, the results are shown in Figure 5. The average particle size of sEVs of MSC is 123.1±4.453nm, and the average particle size of sIVs is 75.28±9.067nm; the particle size of sIVs is smaller than that of sEVs.
4.3等细胞量下sEVs和sIVs的产量对比4.3 Comparison of the yields of sEVs and sIVs at equal cell mass
为了比较两种类型囊泡的产量，我们同时从细胞培养上清液和贴壁细胞中收集sEVs和sIVs。经过NTA检测，结果显示，1×107细胞产生的sIVs囊泡数量比sEVs高10倍至20倍（图6A），来源于1×107细胞的sIVs中的蛋白质产物比sEVs高20倍至40倍（图6B）。这说明sIVs的产量远高于sEVs。To compare the yield of the two types of vesicles, we collected sEVs and sIVs from cell culture supernatants and adherent cells at the same time. After NTA detection, the results showed that the number of sIVs vesicles produced by 1×107 cells was 10 to 20 times higher than that of sEVs (Figure 6A), and the protein products in sIVs derived from 1×107 cells were 20 to 40 times higher than that of sEVs (Figure 6B). This shows that the yield of sIVs is much higher than that of sEVs.
4.4 sEVs与sIVs的蛋白种类分布差异4.4 Differences in protein distribution between sEVs and sIVs
通过SDS-PAGE将细胞，sEVs和sIVs的全蛋白分离，使用考马斯亮蓝染色显示总蛋白分布。染色结果表明细胞含有的蛋白质显示出最丰富的条带，具有多个高丰度蛋白条带；sEVs含有的蛋白种类较少，高丰度蛋白位于200kD和70kD左右；sIVs含有的蛋白质种类多于sEVs，高丰度蛋白位于250kD和55kD左右（图7）。不同细胞之间的蛋白分布有所差异，这初步说明sEVs和sIVs具有不同的蛋白质成分，并且与细胞和sEVs的总蛋白分布都不同。The whole proteins of cells, sEVs and sIVs were separated by SDS-PAGE, and the total protein distribution was displayed by Coomassie Brilliant Blue staining. The staining results showed that the proteins contained in cells showed the most abundant bands, with multiple high-abundance protein bands; sEVs contained fewer types of proteins, and the high-abundance proteins were located around 200kD and 70kD; sIVs contained more types of proteins than sEVs, and the high-abundance proteins were located around 250kD and 55kD (Figure 7). The protein distribution between different cells was different, which preliminarily indicated that sEVs and sIVs had different protein components and were different from the total protein distribution of cells and sEVs.
为进一步分析细胞及其sEVs和sIVs的蛋白表达特征。使用Western blot检测等蛋白条件下细胞及其sEVs和sIVs的外泌体标志蛋白，Alix，HSP70, TSG101, CD63, CD81的表达情况。结果如图8所示。细胞的sEVs表达最多的外泌体标志蛋白，细胞本身也表达一定量的Alix，HSP70, TSG101, CD63；但是sIVs的Alix和CD81基本不表达，HSP70, TSG101和CD63的表达水平也远低于细胞和sEVs，这进一步说明sIVs不具有外泌体的特征，不是外泌体在细胞内的前体。To further analyze the protein expression characteristics of cells and their sEVs and sIVs. Western blot was used to detect the expression of exosome marker proteins, Alix, HSP70, TSG101, CD63, and CD81 of cells and their sEVs and sIVs under equal protein conditions. The results are shown in Figure 8. The sEVs of cells expressed the most exosome marker proteins, and the cells themselves also expressed a certain amount of Alix, HSP70, TSG101, and CD63; however, Alix and CD81 of sIVs were basically not expressed, and the expression levels of HSP70, TSG101 and CD63 were also much lower than those of cells and sEVs, which further shows that sIVs do not have the characteristics of exosomes and are not the precursors of exosomes in cells.
4.5不同温度下sEVs和sIVs的稳定性对比4.5 Comparison of the stability of sEVs and sIVs at different temperatures
为了评估两种囊泡在不同温度下的稳定性，将sIVs和sEVs悬浮液等分为三部分，并在不同温度（-80°C、4°C和37°C）下储存。24小时后，评估sEVs和sIVs的形态、大小和蛋白质的量。两种囊泡在-80°C和4°C下都是稳定的。然而，透射电镜图像显示，sEVs的形态在37℃时受损，具有不规则的形状、破碎的囊泡和粗糙的边界（图9），并且sEVs的数量也减少（图10B），而sIVs在37℃下的形态和颗粒数量都保持稳定。以上结果表明，sIVs比sEVs具有更高的热稳定性。To evaluate the stability of the two vesicles at different temperatures, the sIVs and sEVs suspensions were equally divided into three parts and stored at different temperatures (-80 ° C, 4 ° C, and 37 ° C). After 24 hours, the morphology, size, and protein amount of sEVs and sIVs were evaluated. Both vesicles were stable at -80 ° C and 4 ° C. However, transmission electron microscopy images showed that the morphology of sEVs was impaired at 37 ° C, with irregular shapes, broken vesicles, and rough boundaries (Figure 9), and the number of sEVs was also reduced (Figure 10B), while the morphology and particle number of sIVs remained stable at 37 ° C. The above results indicate that sIVs have higher thermal stability than sEVs.
4.6超分辨成像显示sIVs在细胞中的分布区别于sEVs4.6 Super-resolution imaging shows that the distribution of sIVs in cells is different from that of sEVs
为深入了解sIVs在细胞内的分布状态，我们通过蛋白质组学分析鉴定了sIVs相较于sEVs独特表达的蛋白，即IV特征蛋白，使该特征蛋白携带绿色荧光蛋白，并进行细胞内成像，目的是可视化sIVs在细胞内的形态。通过对sIVs和sEVs进行蛋白质组学分析，鉴定出sIVs中独特表达的蛋白质。将蛋白的表达丰度由高到低排序，并显示前50种蛋白（图11）。我们观察到，在MSCs中TMEM214蛋白的表达丰度最高。TMEM214是一种参与囊泡运输和蛋白质运输等细胞过程的跨膜蛋白质（Zhao J., Xu J., Wang Y., et al. Membrane LocalizedGbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt.Plants (Basel). 2022 Sep 8;11(18):2342.）。因此，我们采用绿色荧光蛋白GFP标记TMEM214，以直观显示sIVs在细胞内的状态，同时使用GFP标记的CD63作为sEVs在细胞内的标记。To gain a deeper understanding of the intracellular distribution of sIVs, we used proteomic analysis to identify proteins uniquely expressed in sIVs compared to sEVs, namely IV signature proteins, and made the signature proteins carry green fluorescent protein and perform intracellular imaging to visualize the morphology of sIVs in cells. Proteins uniquely expressed in sIVs were identified by proteomic analysis of sIVs and sEVs. The protein expression abundance was ranked from high to low, and the top 50 proteins were displayed (Figure 11). We observed that the expression abundance of TMEM214 protein was the highest in MSCs. TMEM214 is a transmembrane protein involved in cellular processes such as vesicle transport and protein transport (Zhao J., Xu J., Wang Y., et al. Membrane LocalizedGbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt.Plants (Basel). 2022 Sep 8;11(18):2342.). Therefore, we used green fluorescent protein GFP to mark TMEM214 to visually display the status of sIVs in cells, and used GFP-labeled CD63 as a marker for sEVs in cells.
超分辨显微镜与全内反射荧光结构照明显微镜（Total Internal ReflectionFluorescence Microscopy combined with Structured Illumination Microscopy ,TIRF-SIM）显示，CD63可见位于细胞膜上(图12A，绿色)，TMEM214未在细胞膜上表达（图12A，红色）。这一发现排除了sIVs源自细胞膜重构的可能性，而且证实了sIVs的起源和活动场所位于细胞内。采用Wildfield-2DSM扫描模式的超分辨显微镜提供了整个细胞蛋白质表达的概述，图像显示CD63和TMEM214在细胞内均存在显著表达（图12B）。随后，在宽场条件下，我们观察了活细胞中这些蛋白标记结构的动态变化。每隔10秒拍照一次，连续拍照15分钟，形成动态视频。在视频截图中，可以观察到sEVs从细胞膜动态释放至细胞外的过程（图12C，绿色，白色箭头），而TMEM214标记的sIVs在细胞内弥散分布，没有被释放到细胞外（图12C，红色）。直观的显微成像表明sIVs在细胞内呈云雾状弥散分布，未被释放到细胞外。Super-resolution microscopy combined with total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) showed that CD63 was visible on the cell membrane (Figure 12A, green), and TMEM214 was not expressed on the cell membrane (Figure 12A, red). This finding excludes the possibility that sIVs originate from cell membrane reconstruction and confirms that the origin and activity site of sIVs are located inside the cell. Super-resolution microscopy using Wildfield-2DSM scanning mode provides an overview of protein expression throughout the cell, and images show that both CD63 and TMEM214 are significantly expressed in the cell (Figure 12B). Subsequently, under wide-field conditions, we observed the dynamic changes of these protein-labeled structures in living cells. Take pictures every 10 seconds and take pictures continuously for 15 minutes to form a dynamic video. In the video screenshot, we can observe the dynamic release of sEVs from the cell membrane to the extracellular space (Figure 12C, green, white arrows), while TMEM214-labeled sIVs are diffusely distributed in the cell and are not released outside the cell (Figure 12C, red). Intuitive microscopic imaging shows that sIVs are diffusely distributed in the cell in a cloud-like manner and are not released outside the cell.
5、小结5. Summary
在本实施例中，我们以MSCs细胞为例，收集并表征sIVs和sEVs。通过透射电镜和纳米粒度分析发现sIVs尺寸显著小于sEVs；细胞、sEVs和sIVs的总蛋白表达模式各不相同，sIVs低表达外泌体标志蛋白；等细胞个数下，sIVs产量显著多于sEVs。在-80℃条件下，sIVs和sEVs稳定性相当，但是37℃条件下，sIVs稳定性显著优于sEVs。通过超分辨成像观察到sEVs经细胞膜释放到细胞外，而sIVs在细胞内进行频繁的活动。总的来说，通过本发明所述方法收集得到的sIVs囊泡，其含有独特的蛋白质构成，在生理温度下稳定性较高，产量远高于细胞外囊泡。In this example, we took MSCs cells as an example to collect and characterize sIVs and sEVs. Through transmission electron microscopy and nanoparticle size analysis, it was found that the size of sIVs was significantly smaller than that of sEVs; the total protein expression patterns of cells, sEVs and sIVs were different, and sIVs expressed low exosome marker proteins; under the same number of cells, the yield of sIVs was significantly more than that of sEVs. Under -80°C conditions, the stability of sIVs and sEVs was comparable, but under 37°C conditions, the stability of sIVs was significantly better than that of sEVs. Super-resolution imaging was used to observe that sEVs were released to the outside of the cell through the cell membrane, while sIVs were frequently active inside the cell. In general, the sIVs vesicles collected by the method described in the present invention contain a unique protein composition, are highly stable at physiological temperature, and have a yield much higher than that of extracellular vesicles.
实施例5：定量蛋白组学分析显示sIVs具有独特的蛋白质表达谱Example 5: Quantitative proteomic analysis shows that sIVs have unique protein expression profiles
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表3实验试剂Table 3 Experimental reagents
1.2实验仪器1.2 Experimental instruments
表4实验仪器Table 4 Experimental instruments
2、实验方法2. Experimental methods
2.1细胞及其sEVs、slVs的蛋白质谱样品制备2.1 Sample preparation for protein profiling of cells and their sEVs and sLVs
2.1.1蛋白提取2.1.1 Protein extraction
1）向细胞、sEVs、sIVs（实施例2制备）中分别加入220ul尿素裂解液（8M尿素，50mMNH4HCO3，蛋白酶抑制剂），室温裂解5分钟。1) 220ul of urea lysis buffer (8M urea, 50mM NH4 HCO3 , protease inhibitor) was added to cells, sEVs, and sIVs (prepared in Example 2) respectively, and lysed at room temperature for 5 minutes.
2）冰上超声破碎（能量35%，ON 3S，OFF 3S，超声时间共计2min），样品管插入冰盒中，离心温度设定为20℃，14,000g 离心10min，取上清，再重复离心一次。2) Ultrasonic disruption on ice (energy 35%, ON 3S, OFF 3S, total ultrasonic time 2min), insert the sample tube into the ice box, set the centrifuge temperature to 20℃, centrifuge at 14,000g for 10min, take the supernatant, and repeat the centrifugation once.
3）BCA法检测蛋白浓度，细胞、sEVs、sIVs每个取100μg蛋白。3) The protein concentration was detected by BCA method, and 100 μg protein was taken from each cell, sEVs, and sIVs.
2.1.2蛋白变性2.1.2 Protein denaturation
1）在细胞、sEVs和sIVs的各个样品管中加入DTT，使其终浓度为10mM，37℃孵育1小时以还原蛋白。1) Add DTT to each sample tube of cells, sEVs, and sIVs to a final concentration of 10 mM and incubate at 37°C for 1 hour to reduce the proteins.
2）在细胞、sEVs和sIVs的各个样品管加入IAA，使其终浓度为40mM，避光下室温孵育1小时。2) Add IAA to each sample tube of cells, sEVs, and sIVs to a final concentration of 40 mM and incubate at room temperature for 1 hour in the dark.
3）首先在集合管上标记上述样本的编号，用HPLC级甲醇平衡10kDa超滤管2次，每次甲醇的体积为150μl，14,000g 5min，再加入300μl 50mM NH4HCO3，随后冲洗两次，加入100μg还原烷基化后的蛋白样本，4℃ 14000g 离心20min，加入300μl 浓度为50mM NH4HCO3清洗三次，更换新的收集管，加入75μl 浓度为50mM NH4HCO3到超滤管中。3) First, mark the number of the above samples on the collection tube, equilibrate the 10kDa ultrafiltration tube twice with HPLC-grade methanol, each time with a volume of 150μl of methanol, 14,000g for 5min, then add 300μl of 50mMNH4HCO3, then rinse twice, add 100μg of the reduced alkylated protein sample, centrifuge at 14000g for 20min at 4℃, add 300μl of 50mMNH4HCO3 and rinse threetimes , replace a new collection tube, and add 75μl of 50mMNH4HCO3 to the ultrafiltrationtube .
2.1.3蛋白酶切2.1.3 Protease cleavage
1）加入3μg质谱用胰酶，37℃孵箱中孵育14小时-16小时。1) Add 3 μg of trypsin for mass spectrometry and incubate in a 37°C incubator for 14-16 hours.
2）第二天，在4℃，14,000g下离心20分钟，加体积为50μl 浓度为50mM NH4HCO3，冲洗2次，向收集管中加入1%（体积比）的甲酸以终止酶切，60℃真空蒸干。2) The next day, centrifuge at 14,000 g for 20 minutes at 4°C, add 50 μl of 50 mM NH4 HCO3 , rinse twice, add 1% (volume ratio) formic acid to the collection tube to terminate the enzyme digestion, and evaporate to dryness at 60°C in a vacuum.
2.1.4建库分馏2.1.4 Library construction and fractionation
1）将30μl的0.1%甲酸水溶液用于重悬样本，并使用nanodrop测量浓度。接着，从每个样本中取出大约10μg的肽段，将它们合并成一个样本S。1) Resuspend the samples in 30 μl of 0.1% formic acid in water and measure the concentration using a nanodrop. Next, take approximately 10 μg of peptides from each sample and combine them into one sample S.
2）从样本S中取出6μg进行DDA模式的质谱分析，而将剩余的S样本进行自制的高PH反相柱的分馏。2) 6 μg of sample S was taken out for mass spectrometry analysis in DDA mode, and the remaining S sample was fractionated on a homemade high pH reverse phase column.
3）为了进行分馏，需按照表5配制所需试剂。Buffer A为100%乙腈（ACN），而BufferB为0.1%的三氟乙酸（TFA）。3) For fractionation, prepare the required reagents according to Table 5. Buffer A is 100% acetonitrile (ACN), and Buffer B is 0.1% trifluoroacetic acid (TFA).
表5 分馏试剂配比Table 5 Fractionation reagent ratio
4）使用铁丝将一层C18膜装入200微升的枪头中。接着，用200μl的Buffer A溶解30毫克的耐高pH的C18填料，并将该填料加入分馏塔。然后，在4℃下以3,000g离心2分钟，加入200μl的Buffer A清洗一次，再加入200μl的Buffer B清洗三次。最后，将柱子留作备用。4) Use a wire to load a layer of C18 membrane into a 200 μl pipette tip. Next, dissolve 30 mg of high pH resistant C18 filler in 200 μl of Buffer A and add the filler to the fractionation column. Then, centrifuge at 3,000 g for 2 minutes at 4°C, add 200 μl of Buffer A to wash once, and then add 200 μl of Buffer B to wash three times. Finally, set the column aside for later use.
5）将酶切后的肽段加载到分馏柱上，并重复加载5次。5) Load the cleaved peptides onto the fractionation column and repeat the loading 5 times.
6）使用200μl的Buffer B清洗分馏柱3次。接着，加入150μl不同浓度的洗脱液，并按梯度进行洗脱。将6%和35%的馏分合并为一个馏分，并从每个馏分中取出1.5μg的肽段进行DDA模式的质谱分析。6) Wash the fractionation column three times with 200 μl of Buffer B. Then, add 150 μl of elution buffer of different concentrations and perform gradient elution. Combine the 6% and 35% fractions into one fraction, and take 1.5 μg of peptides from each fraction for mass spectrometry analysis in DDA mode.
2.2细胞及其sEVs、sIVs液相质谱参数2.2 HPLC-MS parameters of cells and their sEVs and sIVs
经过酶切处理的肽段，使用A相（含有0.1%甲酸、2%乙腈和97.9%水）以3μl/分钟的流速，被加载到自制的Trap柱（规格为100微米×2厘米，C18填料，粒径3微米，120A）。随后，利用不同梯度的B相（含有97.9%乙腈、2%水和0.1%甲酸）对Trap柱进行洗脱。这些洗脱出的肽段经过分析柱（规格为150微米×15厘米，C18填料，粒径1.9微米，120A）形成带电喷雾，最终进入质谱检测器。The peptides after enzyme digestion were loaded onto a homemade Trap column (100 μm × 2 cm, C18 filler, 3 μm particle size, 120A) using phase A (containing 0.1% formic acid, 2% acetonitrile and 97.9% water) at a flow rate of 3 μl/min. Subsequently, the Trap column was eluted using different gradients of phase B (containing 97.9% acetonitrile, 2% water and 0.1% formic acid). These eluted peptides passed through an analytical column (150 μm × 15 cm, C18 filler, 1.9 μm particle size, 120A) to form an electrospray and finally entered the mass spectrometer detector.
B相的梯度设置如下：0分钟至5%，2分钟至10%，65分钟至22%，91分钟至35%，92分钟至80%，105分钟至80%，106分钟至5%，120分钟至5%，整个过程的流速维持在500纳升/分钟。The gradient of phase B was set as follows: 0 min to 5%, 2 min to 10%, 65 min to 22%, 91 min to 35%, 92 min to 80%, 105 min to 80%, 106 min to 5%, 120 min to 5%, and the flow rate was maintained at 500 nL/min throughout the process.
在进行DDA扫描时，质谱参数设定如下：TOF MS的累加时间为0.25秒，质量扫描范围覆盖300-1500道尔顿（Da），仅检测+2至+5价的离子，质量偏差需小于50ppm。每个循环内最多监测60个离子，每次检测后，将已检测的离子隔离16秒。碎裂能量模式采用动态碎裂模式。Product ion的累加时间为0.04秒，并采用高灵敏扫描模式。When performing DDA scanning, the mass spectrometry parameters were set as follows: the accumulation time of TOF MS was 0.25 seconds, the mass scanning range covered 300-1500 Daltons (Da), only ions with valences of +2 to +5 were detected, and the mass deviation was required to be less than 50 ppm. A maximum of 60 ions were monitored in each cycle, and after each detection, the detected ions were isolated for 16 seconds. The fragmentation energy mode used the dynamic fragmentation mode. The accumulation time of the Product ion was 0.04 seconds, and the high-sensitivity scanning mode was used.
而在进行DIA扫描时，质谱参数则有所不同：TOF MS的累加时间为0.05秒，二级扫描采用高灵敏模式。可变窗口数设定为100个，每个窗口的累加时间为30毫秒，质量扫描范围同样为300-1500Da。使用SWATH Variable Window Calculator_V1.1程序计算每个可变窗口的具体质量范围。When performing DIA scanning, the mass spectrometry parameters are different: the accumulation time of TOF MS is 0.05 seconds, and the secondary scan uses high sensitivity mode. The number of variable windows is set to 100, the accumulation time of each window is 30 milliseconds, and the mass scanning range is also 300-1500Da. The specific mass range of each variable window is calculated using the SWATH Variable Window Calculator_V1.1 program.
2.3细胞及其sEVs、sIVs蛋白质谱数据处理及生物信息学分析2.3 Cell and sEVs, sIVs protein spectrum data processing and bioinformatics analysis
使用Proteinpilot软件（版本5.0.1）对DDA模式所收集的原始数据进行数据库搜索，以trypsin作为酶切方式。所选用的数据库为Uniprot人数据库，其中包含20431个已注释的蛋白，发布于2019年7月。筛选条件为unused protScore大于0.05。将Proteinpilot的搜索结果导入SWATH软件（版本2.0）作为数据库，以便对DIA模式收集的数据进行定量。The raw data collected in DDA mode were searched using Proteinpilot software (version 5.0.1) with trypsin as the restriction enzyme. The database used was the Uniprot database, which contains 20,431 annotated proteins and was published in July 2019. The screening condition was unused protScore greater than 0.05. The search results of Proteinpilot were imported into SWATH software (version 2.0) as a database to quantify the data collected in DIA mode.
在定量过程中，每个蛋白选取6个肽段，每个肽段再选择6个transitions（离子对）。肽段的confidence设定为99%，FDR（假阳性率）设定为1%。同时，排除修饰肽段，峰提取窗口设为10分钟，质量偏差控制在50ppm以内。每10分钟选取2个内源性肽段来校正保留时间，并将输出的峰面积作为定量值。During the quantitative process, 6 peptides were selected for each protein, and 6 transitions (ion pairs) were selected for each peptide. The confidence of the peptide was set to 99%, and the FDR (false positive rate) was set to 1%. At the same time, modified peptides were excluded, the peak extraction window was set to 10 minutes, and the mass deviation was controlled within 50ppm. Two endogenous peptides were selected every 10 minutes to correct the retention time, and the output peak area was used as the quantitative value.
对蛋白表达数据的处理涉及以下步骤：首先对原始定量值进行log2转换以满足正态分布，然后使用R语言中的preprocessCore包中的normalize.quantiles函数进行归一化。去除没有基因名称的蛋白后，使用R语言stats包进行差异分析，筛选出P值小于0.05且变化倍数大于1.5倍的蛋白作为差异蛋白。同时，校正后的p值设置为小于0.05。The processing of protein expression data involves the following steps: first, the original quantitative values are log2 transformed to meet the normal distribution, and then normalized using the normalize.quantiles function in the preprocessCore package in the R language. After removing proteins without gene names, the stats package in the R language is used for differential analysis, and proteins with a P value less than 0.05 and a change fold greater than 1.5 are screened as differential proteins. At the same time, the corrected p value is set to less than 0.05.
GO和通路分析则通过Cytoscape插件clueGo进行。在GO富集分析中，选择细胞组分（CC）、分子功能（MF）和生物过程（BP）进行分析。而在通路富集分析中，选择kegg和reactome的数据库进行。GO and pathway analysis were performed using the Cytoscape plug-in clueGo. In GO enrichment analysis, cell component (CC), molecular function (MF), and biological process (BP) were selected for analysis. In pathway enrichment analysis, the kegg and reactome databases were selected for analysis.
Heatmap、主成分分析（PCA）评分、Venn图、火山图等使用R语言进行或者Hiplot软件进行绘制。京都基因和基因组百科全书（KEGG）途径富集分析使用Metascape在线分析软件。Heatmap, principal component analysis (PCA) score, Venn diagram, volcano map, etc. were performed using R language or drawn using Hiplot software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using Metascape online analysis software.
2.4酶联免疫吸附实验2.4 ELISA
收集MSCs的sEVs和sIVs，调整至等质量，按照酶联免疫吸附实验（Enzyme linkedimmunosorbent assay, ELISA）试剂盒的说明书进行操作。将样品加入包被有抗体的孔中，孵育2小时，清洗孔板；加入生物素，孵育1小时，清洗孔板；加入HRP，孵育1小时，清洗孔板；加入TMB底物，孵育20分钟；加入STOP液，直至颜色明显。使用酶标仪读取孔板中每个孔的吸光度值，波长使用450nm/540nm。Collect sEVs and sIVs of MSCs, adjust to equal mass, and operate according to the instructions of the enzyme linked immunosorbent assay (ELISA) kit. Add the sample to the wells coated with antibodies, incubate for 2 hours, and wash the wells; add biotin, incubate for 1 hour, wash the wells; add HRP, incubate for 1 hour, wash the wells; add TMB substrate, incubate for 20 minutes; add STOP solution until the color is obvious. Use a microplate reader to read the absorbance value of each well in the well plate, using a wavelength of 450nm/540nm.
3、统计学处理3. Statistical processing
实验数据以均数±标准差（）表示。实验数据均进行正态检验。使用SPSS22.0分析所有定量数据，两组之间使用t test进行统计学分析。P值<0.05为统计学显著差异。The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. SPSS22.0 was used to analyze all quantitative data, and t test was used for statistical analysis between the two groups.P value < 0.05 was considered statistically significant.
4、实验结果4. Experimental results
4.1韦恩图显示细胞及其sEVs、sIVs蛋白成分差异4.1 Venn diagram showing differences in protein composition of cells and their sEVs and sIVs
为了表征sEVs和sIVs的分子组成，我们采用无标记质谱技术对来源于MSCs细胞的sEVs和sIVs进行了蛋白质组学分析，并且与细胞进行对比分析。在MSCs细胞中，识别出2744种蛋白质；同时，在sEVs中分别检测到1678种蛋白质，在sIVs中发现2066种蛋白质（图13）。它们之间的蛋白质种类存在一定重合，但是并不完全相同。sIVs中的蛋白质含量比sEVs中的更为多样。In order to characterize the molecular composition of sEVs and sIVs, we used label-free mass spectrometry to perform proteomic analysis of sEVs and sIVs derived from MSCs cells and compared them with cells. In MSCs cells, 2744 proteins were identified; at the same time, 1678 proteins were detected in sEVs and 2066 proteins were found in sIVs (Figure 13). There is some overlap in the types of proteins between them, but they are not exactly the same. The protein content in sIVs is more diverse than that in sEVs.
4.2主成分分析显示细胞及其sEVs、sIVs蛋白成分差异4.2 Principal component analysis reveals differences in protein composition of cells and their sEVs and sIVs
通过PCA分析细胞及其sEVs，sIVs的蛋白质组分（图14），结果表明细胞、sEVs和sIVs表现出不同的蛋白质分布模式。sEVs和sIVs在蛋白质表达上表现出显著的差异。这说明sIVs区别于sEVs，具有独特的蛋白表达特征。PCA analysis of the protein components of cells and their sEVs and sIVs (Figure 14) showed that cells, sEVs and sIVs showed different protein distribution patterns. sEVs and sIVs showed significant differences in protein expression. This indicates that sIVs are different from sEVs and have unique protein expression characteristics.
4.3细胞及其sEVs、sIVs的可定量蛋白差异分析4.3 Quantifiable protein differential analysis of cells and their sEVs and sIVs
进一步统计表明，在MSC细胞中，sEVs和sIVs之间有1425种差异表达蛋白，其中sIVs相较于sEVs显著下调的有753个，显著上调的有672个，其中差异倍数大于10倍的有468，sIVs相较于sEVs显著下调的有227个，显著上调的有241个（图15）。进一步体现了sIVs的独特性。Further statistics showed that there were 1425 differentially expressed proteins between sEVs and sIVs in MSC cells, of which 753 were significantly downregulated in sIVs compared with sEVs, 672 were significantly upregulated, 468 of which had a difference of more than 10 times, 227 were significantly downregulated in sIVs compared with sEVs, and 241 were significantly upregulated (Figure 15), further demonstrating the uniqueness of sIVs.
sEVs和sIVs中表达上调和下调最显著的蛋白质并不完全相同（图16）。然而，膜相关蛋白在sIVs中的表达水平较低，而内质网和核糖体相关蛋白在sIVs中更丰富。这初步表明sIVs是由细胞产生的独特实体，而不是细胞裂解物或细胞外囊泡的前体或片段。The most significantly upregulated and downregulated proteins in sEVs and sIVs were not exactly the same (Figure 16). However, membrane-associated proteins were expressed at lower levels in sIVs, while endoplasmic reticulum and ribosome-associated proteins were more abundant in sIVs. This initially suggests that sIVs are unique entities produced by cells, rather than precursors or fragments of cell lysates or extracellular vesicles.
4.4细胞及其sEVs、sIVs的外泌体标志物表达差异4.4 Differential expression of exosomal markers in cells and their sEVs and sIVs
我们提取了MISEV2018推荐的外泌体标记物列表（Théry C., Witwer K. W.,Aikawa E., et al. Minimal information for studies of extracellular vesicles2018 (MISEV2018): a position statement of the International Society forExtracellular Vesicles and update of the MISEV2014 guidelines [J]. Journal ofextracellular vesicles, 2018, 7(1): 1535750.），并将sEVs和sIVs与这些标记物进行了比较。sEVs表现出更高的外泌体标记物表达水平，而sIVs对大多数外泌体标志蛋白的表达水平都较低（图17）。这进一步说明sIVs不具有外泌体特征，sIVs是来自于细胞内部的独特囊泡。We extracted the list of exosome markers recommended by MISEV2018 (Théry C., Witwer K. W., Aikawa E., et al. Minimal information for studies of extracellular vesicles2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines [J]. Journal of extracellular vesicles, 2018, 7(1): 1535750.) and compared sEVs and sIVs with these markers. sEVs showed higher expression levels of exosome markers, while sIVs had lower expression levels of most exosome marker proteins (Figure 17). This further suggests that sIVs do not have exosome characteristics and are unique vesicles from the inside of cells.
4.5细胞及其sEVs、sIVs的细胞器标志蛋白表达差异4.5 Differential expression of organelle marker proteins in cells and their sEVs and sIVs
我们还对sEVs和sIVs的细胞器蛋白质表达谱进行了比较分析。总体而言，sIVs所含的细胞内细胞器蛋白质的水平高于sEVs（图18）。特别是，sIVs显示出与膜富集的细胞器（如内体、内质网和高尔基体）相关的蛋白质表达水平升高。相比之下，sEVs含有更丰富的细胞膜蛋白质（图18）。这说明sIVs具有细胞内的特征。We also performed a comparative analysis of the organelle protein expression profiles of sEVs and sIVs. Overall, sIVs contained higher levels of intracellular organelle proteins than sEVs (Figure 18). In particular, sIVs showed elevated expression levels of proteins associated with membrane-enriched organelles such as endosomes, endoplasmic reticulum, and Golgi apparatus. In contrast, sEVs contained more abundant cellular membrane proteins (Figure 18). This suggests that sIVs have intracellular characteristics.
4.6细胞及其sEVs、sIVs的Clathrin蛋白家族表达差异4.6 Differential expression of Clathrin protein family in cells and their sEVs and sIVs
在细胞内囊泡所含的蛋白质中，Clathrin蛋白家族对于囊泡的组织和活性至关重要，Clathrin蛋白通过促进分泌和内吞途径中内质网、高尔基体和内体等细胞器之间的货物转运，在细胞内转运中发挥关键作用。鉴于网格蛋白家族的重要作用，我们比较了sEVs和sIVs中网格蛋白家族蛋白的表达水平。值得注意的是，我们观察到大多数网格蛋白家族蛋白在sIVs中的表达上调，而sEVs则呈现低表达（图19）。这一发现表明，我们分离出的sIVs可能在细胞内部参与不同细胞隔室之间的交流。Among the proteins contained in intracellular vesicles, the Clathrin protein family is essential for the organization and activity of vesicles. Clathrin proteins play a key role in intracellular transport by facilitating cargo transport between organelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes in the secretory and endocytic pathways. Given the important role of the clathrin family, we compared the expression levels of clathrin family proteins in sEVs and sIVs. Notably, we observed that most clathrin family proteins were upregulated in sIVs, while sEVs showed low expression (Figure 19). This finding suggests that the sIVs we isolated may participate in the communication between different cellular compartments inside the cell.
4.7 sIVs独有蛋白的富集分析4.7 Enrichment analysis of sIVs-specific proteins
实施例4中，我们分析得到了sIVs区别于sEVs表达的蛋白质106种，这些蛋白是sIVs表达的独特蛋白，可以代表sIVs的特征，我们对这些蛋白质进行了基因富集分析。就细胞成分（CC）而言，这些蛋白质与COPII包被的内质网至高尔基体运输囊泡、运输囊泡，包被囊泡，内质网至高尔基体运输囊膜，内质网至Golgi中间区室，运输囊泡膜，包被泡膜等相关（图20）。同时，在生物过程（BP）类别中，术语为甘油磷脂生物合成过程、对内质网应激的反应、泛素依赖性ERAD途径、细胞内蛋白质转运和内质网到高尔基体小泡介导的转运等（图21）。这一结果表明我们分离得到的sIVs是细胞内固有的囊泡成分。In Example 4, we analyzed and obtained 106 proteins that are different from sEVs in expression. These proteins are unique proteins expressed by sIVs and can represent the characteristics of sIVs. We performed gene enrichment analysis on these proteins. In terms of cellular components (CC), these proteins are related to COPII-coated endoplasmic reticulum to Golgi transport vesicles, transport vesicles, coated vesicles, endoplasmic reticulum to Golgi transport vesicle membranes, endoplasmic reticulum to Golgi intermediate compartments, transport vesicle membranes, coated vesicle membranes, etc. (Figure 20). At the same time, in the biological process (BP) category, the terms are glycerophospholipid biosynthesis process, response to endoplasmic reticulum stress, ubiquitin-dependent ERAD pathway, intracellular protein transport, and endoplasmic reticulum to Golgi vesicle-mediated transport, etc. (Figure 21). This result shows that the sIVs we isolated are intrinsic vesicle components in cells.
4.8 sEVs和sIVs含有的细胞因子对比4.8 Comparison of cytokines contained in sEVs and sIVs
细胞内囊泡参与各种分泌因子的细胞内转运，而外泌体则将这些因子携带到细胞外。因此，我们比较了sEVs和sIVs携带的细胞因子水平。蛋白质组学分析显示，与sEVs相比，sIVs中白介素-1β（Interleukin, IL-1β）和类胰岛素生长因子2（Insulin-like GrowthFactor 1, IGF-2）水平较低（图22）。随后，我们采用ELISA进一步量化低丰度细胞因子。结果表明，在相同质量的基础上，sIVs含有高水平的类胰岛素生长因子1（Insulin-likeGrowth Factor 1, IGF-1）、表皮生长因子（Epidermal Growth Factor, EGF）和白介素-10（Interleukin, IL-10）（图23）。同时，sEVs和sIVs之间IL-6（图23D）水平无显著差异，sIVs中肿瘤坏死因子α（Tumor Necrosis Factor α, TNFα）（图23E）水平较低。Intracellular vesicles are involved in the intracellular transport of various secretory factors, while exosomes carry these factors to the extracellular space. Therefore, we compared the levels of cytokines carried by sEVs and sIVs. Proteomic analysis showed that sIVs had lower levels of interleukin-1β (IL-1β) and insulin-like growth factor 2 (IGF-2) compared with sEVs (Figure 22). Subsequently, we used ELISA to further quantify low-abundance cytokines. The results showed that on the basis of the same mass, sIVs contained high levels of insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and interleukin-10 (IL-10) (Figure 23). Meanwhile, there was no significant difference in IL-6 levels between sEVs and sIVs ( Figure 23D ), and the level of Tumor Necrosis Factor α (TNFα) ( Figure 23E ) was lower in sIVs.
5、小结5. Summary
在本实施例中，我们以MSCs细胞为例，应用蛋白质组学技术表征了细胞、sEVs和sIVs的蛋白质组成。结果表明，sIVs具有独特的蛋白质表达谱，区别于细胞和sEVs；sIVs低表达外泌体标志物，高表达细胞内富膜细胞器的标志蛋白和Clathrin蛋白家族，这表明sIVs承担细胞内物质运输的角色，并且介导了细胞内细胞器交流；对sIVs的基因富集分析直接提示sIVs涉及内质网和高尔基体间物质转运，这包括内质网至高尔基体囊泡介导的正向转运以及从高尔基体返回内质体的逆行囊泡介导的转运。这些发现强烈暗示sIVs在细胞内物质转运过程中发挥着关键作用，特别是介导细胞器间的物质交流。其中，COP包被的囊泡被广泛报道参与内质网起始的细胞内物质转运过程。在此过程中，内质网中正确折叠和装配的蛋白质被封装成COP包被的转运囊泡，随后这些囊泡从内质网膜上脱离。紧接着，囊泡脱掉包被并相互融合，形成囊泡管状簇。高尔基体则负责对这些从内质网接收的蛋白质和脂质进行修饰，并将它们分配到细胞膜、内体和分泌囊泡中。蛋白质和脂质在高尔基体内按顺式到反面的方向移动，并通过囊泡运输完成这一过程。通过蛋白组学分析，进一步证实sIVs参与了细胞内的囊泡运输过程，与内质网、高尔基体和COP包被的囊泡密切相关。In this example, we used MSCs cells as an example and used proteomics technology to characterize the protein composition of cells, sEVs and sIVs. The results showed that sIVs had a unique protein expression profile, which was different from cells and sEVs; sIVs expressed low exosome markers and highly expressed marker proteins and Clathrin protein family of intracellular membrane-rich organelles, which indicated that sIVs played a role in intracellular material transport and mediated intracellular organelle communication; gene enrichment analysis of sIVs directly suggested that sIVs were involved in the transport of substances between the endoplasmic reticulum and the Golgi apparatus, including forward transport mediated by endoplasmic reticulum to Golgi apparatus vesicles and retrograde vesicle-mediated transport from the Golgi apparatus back to the endoplasm. These findings strongly suggest that sIVs play a key role in the intracellular material transport process, especially mediating the exchange of substances between organelles. Among them, COP-coated vesicles have been widely reported to participate in the intracellular material transport process initiated by the endoplasmic reticulum. During this process, correctly folded and assembled proteins in the endoplasmic reticulum are encapsulated into COP-coated transport vesicles, which then detach from the endoplasmic reticulum membrane. Next, the vesicles shed their coating and fuse with each other to form tubular clusters of vesicles. The Golgi apparatus is responsible for modifying these proteins and lipids received from the endoplasmic reticulum and distributing them to the cell membrane, endosomes, and secretory vesicles. Proteins and lipids move in the cis-to-trans direction within the Golgi apparatus and complete this process through vesicular transport. Proteomic analysis further confirmed that sIVs is involved in the intracellular vesicular transport process and is closely related to the endoplasmic reticulum, Golgi apparatus, and COP-coated vesicles.
以上结果表明sIVs与sEVs截然不同，sIVs在细胞内物质运输中发挥重要作用，sIVs是一种独特的囊泡群。The above results indicate that sIVs are completely different from sEVs, sIVs play an important role in intracellular substance transport, and sIVs are a unique group of vesicles.
实施例6：sIVs具有独特的miRNA表达谱Example 6: sIVs have unique miRNA expression profiles
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表6实验试剂Table 6 Experimental reagents
1.2实验仪器1.2 Experimental instruments
表7实验仪器Table 7 Experimental instruments
2、实验方法2. Experimental methods
2.1数据采集及分析2.1 Data collection and analysis
2.1.1 RNA分离、文库制备和测序2.1.1 RNA isolation, library preparation, and sequencing
分离富集sEVs和sIVs（实施例2制备）后使用PBS重悬，在1%琼脂糖凝胶上检测RNA降解和污染。使用NanoPhotometer®分光光度计检查RNA纯度。RNA浓度使用Qubit®2.0Flurometer中的Qubit™RNA测定试剂盒进行测量。使用安捷伦生物分析仪2100系统的RNANano 6000测定试剂盒进行检测。After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS and tested for RNA degradation and contamination on a 1% agarose gel. RNA purity was checked using a NanoPhotometer® spectrophotometer. RNA concentration was measured using the Qubit™ RNA assay kit in a Qubit® 2.0 Flurometer. The RNANano 6000 assay kit for the Agilent Bioanalyzer 2100 system was used for detection.
2.1.2用于小RNA测序的文库制备2.1.2 Library preparation for small RNA sequencing
取每个样品3μg的总RNA量作为小RNA文库的输入样本。测序文库是使用NEBNext®Multiplex 软件的Small RNA Library Prep Set for Illumina®（生成的，并添加了索引代码目的是将序列分配给每个样品。使用LongAmp Taq 2X Master Mix、SR Primer forillumina和index（X）引物在PCR仪器上进行扩增。随后在8%聚丙烯酰胺凝胶（100V，80分钟）上对PCR产物进行纯化。回收对应于140~160bp的DNA片段，并将其溶解在8μL洗脱缓冲液中。最后，使用DNA High Sensitivity Chips在Agilent Bioanalyzer 2100系统上评估文库质量。3 μg of total RNA from each sample was used as the input sample for the small RNA library. The sequencing library was generated using the Small RNA Library Prep Set for Illumina® (of NEBNext® Multiplex software, and an index code was added to assign the sequence to each sample. Amplification was performed on a PCR instrument using LongAmp Taq 2X Master Mix, SR Primer for Illumina, and index (X) primers. The PCR product was then purified on an 8% polyacrylamide gel (100 V, 80 min). DNA fragments corresponding to 140-160 bp were recovered and dissolved in 8 μL elution buffer. Finally, the library quality was assessed on an Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.
2.1.3簇生成和测序2.1.3 Cluster generation and sequencing
在cBot Cluster Generation System上根据制造商的说明使用TruSeq SRCluster Kit v3-cBot-HS（Illumia）对索引编码的样品进行簇生成。簇生成后，在IlluminaHiseq 2500/2000平台上进行测序，生成50bp单端读数以制备文库。The index-coded samples were clustered using the TruSeq SRCluster Kit v3-cBot-HS (Illumia) on the cBot Cluster Generation System according to the manufacturer’s instructions. After cluster generation, sequencing was performed on the Illumina HiSeq 2500/2000 platform to generate 50 bp single-end reads for library preparation.
2.1.4数据分析2.1.4 Data Analysis
1) 质量控制1) Quality Control
首先，通过自定义的Perl和Python脚本处理fastq格式的原始数据（raw reads）。在此步骤中，通过去除含有ploy-N、5’端接头污染、无3’端接头或插入标签、含有ploy A或T或G或C以及低质量的读数，从原始数据中获得清洁数据（clean reads）。同时，计算原始数据的Q20、Q30和GC含量。然后，从清洁读数中选择一定长度范围进行所有下游分析。使用Bowtie（Langmead B., Trapnell C., Pop M., Salzberg S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome [J]. Genomebiology, 2009, 10(3): R25.）将小RNA标签映射到参考序列，不允许错配，以分析其在参考序列上的表达和分布。First, the raw data (raw reads) in fastq format were processed by custom Perl and Python scripts. In this step, clean reads were obtained from the raw data by removing reads containing ploy-N, 5' end adapter contamination, no 3' end adapter or inserted tags, containing ploy A or T or G or C, and low quality. At the same time, the Q20, Q30 and GC content of the raw data were calculated. Then, a certain length range was selected from the clean reads for all downstream analyses. Bowtie (Langmead B., Trapnell C., Pop M., Salzberg S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome [J]. Genomebiology, 2009, 10(3): R25.) was used to map the small RNA tags to the reference sequence, without allowing mismatches, to analyze their expression and distribution on the reference sequence.
2) 已知miRNA比对2) Alignment of known miRNAs
将映射的小RNA标签用于寻找已知的miRNA。以miRBase20.0作为参考，使用修改后的软件mirdeep2（Friedländer M. R., Mackowiak S. D., Li N., et al. miRDeep2accurately identifies known and hundreds of novel microRNA genes in sevenanimal clades [J]. Nucleic acids research, 2012, 40(1): 37-52）和srna-tools-cli来获取潜在的miRNA并绘制二级结构。使用自定义脚本分别获取具有特定长度的已识别miRNA的第一个位置上的miRNA计数和碱基偏倚，以及所有已识别miRNA的每个位置上的miRNA计数和碱基偏倚。The mapped small RNA tags were used to search for known miRNAs. Using miRBase20.0 as a reference, the modified software mirdeep2 (Friedländer M. R., Mackowiak S. D., Li N., et al. miRDeep2accurately identifies known and hundreds of novel microRNA genes in sevenanimal clades [J]. Nucleic acids research, 2012, 40(1): 37-52) and srna-tools-cli were used to obtain potential miRNAs and draw secondary structures. Custom scripts were used to obtain the miRNA counts and base biases at the first position of identified miRNAs with a specific length, as well as the miRNA counts and base biases at each position of all identified miRNAs.
3) 小RNA注释摘要3) Summary of small RNA annotation
总结之前获得的所有比对和注释。在之前的比对和注释中，一些小RNA标签可能被映射到多个类别。为了确保每个唯一的小RNA只映射到一个注释，我们遵循以下优先级规则：已知的miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene> 新miRNA。Summarize all alignments and annotations obtained previously. In the previous alignments and annotations, some small RNA tags may be mapped to multiple categories. To ensure that each unique small RNA is mapped to only one annotation, we follow the following priority rule: known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>new miRNA.
4) 数据分析4) Data Analysis
目标基因预测使用miRanda进行预测，miRNA靶基因预测为miRanda和RNAhybrid两个软件的交集。差异表达的miRNA输入数据为miRNA表达水平分析中得到的readcount数据。对于具有生物学重复性的样品：使用DESeq R包（3.0.3）对两种条件/组进行差异表达分析。使用Benjamini&Hochberg方法对P值进行调整。默认情况下，将校正后的P值设为0.05作为显著差异表达的阈值。热图、主成分分析、韦恩图、火山图等使用Hiplot在线绘制并使用Adobe Illustrator进行调整。Target gene prediction was performed using miRanda, and miRNA target gene prediction was the intersection of miRanda and RNAhybrid. The differentially expressed miRNA input data was the readcount data obtained from the miRNA expression level analysis. For samples with biological repeatability: the DESeq R package (3.0.3) was used to perform differential expression analysis on the two conditions/groups. The P value was adjusted using the Benjamini&Hochberg method. By default, the corrected P value was set to 0.05 as the threshold for significant differential expression. Heat maps, principal component analysis, Venn diagrams, volcano maps, etc. were drawn online using Hiplot and adjusted using Adobe Illustrator.
5) GO和KEGG富集分析5) GO and KEGG enrichment analysis
对差异表达miRNA的目标基因候选物（以下简称“目标基因候选物”）进行GO富集分析。GO富集分析采用基于Wallenius非中心超几何分布的GOseq，可以调整基因长度偏差。使用KOBAS软件来测试目标基因候选物在KEGG通路中的统计富集。GO enrichment analysis was performed on the target gene candidates of differentially expressed miRNAs (hereinafter referred to as "target gene candidates"). GO enrichment analysis used GOseq based on Wallenius non-central hypergeometric distribution, which can adjust gene length bias. KOBAS software was used to test the statistical enrichment of target gene candidates in KEGG pathways.
3、实验结果3. Experimental results
3.1 sEVs和sIVs中的相对RNA丰度3.1 Relative RNA abundance in sEVs and sIVs
首先我们使用生物分析仪分析了sEVs和sIVs的RNA丰度（图24）。First, we analyzed the RNA abundance of sEVs and sIVs using a bioanalyzer ( Figure 24 ).
3.2 sEVs和sIVs中的small RNA分布3.2 Distribution of small RNAs in sEVs and sIVs
细胞及细胞外囊泡相关的small RNA是近几年研究的热点，尤其是 microRNA（miRNA）具有多种生物学功能并可作为多种疾病的生物标志物。我们分析了sEVs和sIVs中的small RNA种类。对各个种类的small RNA与总RNA的比对、注释情况进行总结。由于存在一个sRNA同时比对上几种不同的注释信息的情况，为了使每个unique sRNA有唯一的注释，按照known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>novel miRNA检测的优先级顺序将small RNA分类，计算每种small RNA占总RNA的比例。结果显示，在sEVs中，YRNA是最主要的RNA（YRNA是一类高度保守的小分子非编码RNA（参见，谢玉鑫, 陈天星, 王丽,等. YRNA：在癌症与非癌症中的研究进展[J]. 中华实验外科杂志, 2021, 38(9) : 1844-1848.））；在sIVs中，miRNA是最主要的RNA。MSCs的sEVs中miRNA占比29.15%，sIVs中miRNA占比92.52%。sIVs具有更加丰富的small RNA种类，其中miRNA的含量占比显著多于sEVs（图25）。Small RNAs related to cells and extracellular vesicles have been a hot topic in recent years, especially microRNAs (miRNAs), which have multiple biological functions and can be used as biomarkers for multiple diseases. We analyzed the types of small RNAs in sEVs and sIVs. The alignment and annotation of small RNAs of various types with total RNA were summarized. Since there are cases where one sRNA is aligned with several different annotation information at the same time, in order to make each unique sRNA have a unique annotation, small RNAs were classified according to the priority order of known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>novel miRNA detection, and the proportion of each small RNA in total RNA was calculated. The results showed that in sEVs, YRNA is the most important RNA (YRNA is a class of highly conserved small non-coding RNA (see Xie Yuxin, Chen Tianxing, Wang Li, et al. YRNA: Research Progress in Cancer and Non-Cancer [J]. Chinese Journal of Experimental Surgery, 2021, 38(9): 1844-1848.)); in sIVs, miRNA is the most important RNA. MiRNA accounted for 29.15% in sEVs of MSCs and 92.52% in sIVs. sIVs have more abundant small RNA species, among which the content of miRNA is significantly higher than that of sEVs (Figure 25).
3.3 sEVs和sIVs的总miRNA表达特征3.3 Global miRNA expression characteristics of sEVs and sIVs
MiRNA具有丰富的生物学调控作用，并且在small RNA中占有很大的比例，因此，我们对miRNA开展了后续分析，使用韦恩图分析sEVs和sIVs所含有的miRNA种类，结果表明在sEVs中分别检测到694种miRNA，在sIVs中发现989种miRNA（图26）。它们之间的miRNA种类存在一定重合，但是并不完全相同。MiRNA has a rich biological regulatory role and accounts for a large proportion of small RNA. Therefore, we conducted a follow-up analysis of miRNA and used a Venn diagram to analyze the types of miRNA contained in sEVs and sIVs. The results showed that 694 miRNAs were detected in sEVs and 989 miRNAs were found in sIVs (Figure 26). There is some overlap in the types of miRNAs between them, but they are not exactly the same.
因为sEVs和sIVs含有共同表达的miRNA，因此后续使用主成分分析对比sEVs和sIVs的miRNA表达模式，结果显示两种囊泡含有的miRNA成分存在较大差异，表达模式不相关（图27），进一步说明sIVs区别于sEVs，含有独特的miRNA表达谱。Because sEVs and sIVs contain commonly expressed miRNAs, principal component analysis was subsequently used to compare the miRNA expression patterns of sEVs and sIVs. The results showed that the miRNA components contained in the two vesicles were quite different and the expression patterns were unrelated ( Figure 27 ), further indicating that sIVs are different from sEVs and contain a unique miRNA expression profile.
3.4 sEVs和sIVs中的高丰度miRNA3.4 Highly Abundant miRNAs in sEVs and sIVs
通过对sEVs和sIVs中前10个高丰度miRNA分析发现miR-148a-3p、let-7i-5p在sEVs中均处于较高表达水平；let-7f-5p在sIVs中处于较高表达水平。同时miR-148-3p、miR-21-5p和miR-100-5p等miRNA在sEVs和sIVs中均处于较高表达水平（图28）。By analyzing the top 10 high-abundance miRNAs in sEVs and sIVs, we found that miR-148a-3p and let-7i-5p were expressed at higher levels in sEVs, and let-7f-5p was expressed at higher levels in sIVs. At the same time, miRNAs such as miR-148-3p, miR-21-5p and miR-100-5p were expressed at higher levels in both sEVs and sIVs (Figure 28).
3.5 sEVs和sIVs差异表达miRNA分析3.5 Analysis of differentially expressed miRNAs in sEVs and sIVs
为了进一步对比sEVs和sIVs之间miRNA的差异，我们对sEVs和sIVs进行了差异表达分析。结果显示MSCs中，sEVs和sIVs有70个差异miRNA，其中sIVs相较于sEVs显著下调的有22个，显著上调的有48个（图29-30）。进一步说明sIVs与sEVs不同。In order to further compare the differences in miRNAs between sEVs and sIVs, we performed differential expression analysis on sEVs and sIVs. The results showed that in MSCs, there were 70 differential miRNAs between sEVs and sIVs, of which 22 were significantly downregulated in sIVs compared with sEVs, and 48 were significantly upregulated (Figures 29-30). This further illustrates that sIVs are different from sEVs.
3.6 sEVs和sIVs差异表达miRNA靶基因富集分析3.6 Enrichment analysis of differentially expressed miRNA target genes between sEVs and sIVs
MiRNA发挥生物学作用的途径是调控下游靶基因，因此我们对比得到各组的差异miRNA后，将这些miRNA的靶基因的集合分别进行基因富集分析，包括GO分析和KEGG分析。后面为了表述方便，我们将“差异表达miRNA的靶基因”称为“候选靶基因”。GO富集分析结果显示，MSCs的sEVs和sIVs的候选靶基因与细胞内代谢进程相关，细胞定位为细胞内膜相关的细胞器等，分子功能与蛋白结合和酶代谢反应等相关（图31A）；KEGG通路分析显示，MSCs的sEVs和sIVs的候选靶基因与轴突导向、细胞分化、内吞作用和免疫调节（T细胞受体信号通路，B细胞受体信号通路）等通路相关（图31B）。The way miRNA exerts its biological effects is to regulate downstream target genes. Therefore, after comparing the differential miRNAs in each group, we performed gene enrichment analysis on the target gene sets of these miRNAs, including GO analysis and KEGG analysis. For the convenience of expression, we will refer to "target genes of differentially expressed miRNAs" as "candidate target genes". The results of GO enrichment analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to intracellular metabolic processes, cell localization was intracellular membrane-related organelles, and molecular functions were related to protein binding and enzyme metabolic reactions (Figure 31A); KEGG pathway analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to pathways such as axon guidance, cell differentiation, endocytosis and immune regulation (T cell receptor signaling pathway, B cell receptor signaling pathway) (Figure 31B).
4、小结4. Summary
在本实施例中，我们以MSCs细胞为对象，利用small RNA测序技术对sEVs和sIVs的small RNA组成进行了细致的分析，并深入探讨了miRNA的表达模式。细胞内的miRNA在细胞核中生成后被运输到细胞质，参与靶基因的调控。因此细胞内产生的miRNA首先要执行其生物学功能，通过调控基因表达和参与各种细胞生物学过程，例如细胞增殖、分化和凋亡。许多 miRNA 被发现在不同类型的干细胞中有特异性表达，调控细胞分化和特定细胞系成熟的过程。另外一些 miRNA可能促进或抑制细胞死亡信号通路，从而影响细胞生存和凋亡。它们可通过调控细胞凋亡相关基因，如 BCL2 家族成员，caspase和p53等，来维持细胞稳态。在细胞内，miRNA 可以作为信号通路调节器来调整细胞生物学过程，例如增长因子信号、应对氧化应激和炎症反应等。它们可以靶向特定信号通路中的关键分子，从而影响整个信号传导途径。我们的实验结果表明sIVs中富含丰富的miRNA，由此推测sIVs在调控基因表达、细胞增殖、分化、生长、凋亡和信号通路中可能发挥关键作用，具有很大的应用潜力。研究结果显示，sIVs展现出了独特的miRNA表达谱，与sEVs存在显著差异，且sIVs中的miRNA含量更为丰富。这一发现表明sIVs具有潜在的生物调节作用，并可能促进了细胞内不同细胞器之间的信息交流。通过对sEVs和sIVs中差异表达的miRNA进行候选基因富集分析，我们进一步发现sIVs与细胞内膜样细胞器存在紧密联系。综上所述，sEVs与sIVs在small RNA成分上存在差异，尤其是miRNA。针对miRNA的富集分析进一步证实sIVs在细胞内物质运输中发挥着重要作用。相较于sEVs，sIVs含有更为多样的miRNA，可能具备更丰富的生物学功能。这些发现为深入理解sIVs在细胞生物学中的功能提供了重要线索。In this example, we used MSCs cells as the object, and used small RNA sequencing technology to conduct a detailed analysis of the small RNA composition of sEVs and sIVs, and deeply explored the expression pattern of miRNA. After the miRNA in the cell is generated in the nucleus, it is transported to the cytoplasm and participates in the regulation of target genes. Therefore, the miRNA produced in the cell must first perform its biological function by regulating gene expression and participating in various cell biological processes, such as cell proliferation, differentiation and apoptosis. Many miRNAs have been found to be specifically expressed in different types of stem cells, regulating the process of cell differentiation and maturation of specific cell lines. Other miRNAs may promote or inhibit cell death signaling pathways, thereby affecting cell survival and apoptosis. They can maintain cell homeostasis by regulating apoptosis-related genes, such as BCL2 family members, caspase and p53. In cells, miRNAs can act as signaling pathway regulators to adjust cell biological processes, such as growth factor signals, responses to oxidative stress and inflammatory responses. They can target key molecules in specific signaling pathways, thereby affecting the entire signal transduction pathway. Our experimental results show that sIVs are rich in miRNAs, which suggests that sIVs may play a key role in regulating gene expression, cell proliferation, differentiation, growth, apoptosis and signaling pathways, and have great application potential. The results showed that sIVs showed a unique miRNA expression profile, which was significantly different from sEVs, and the miRNA content in sIVs was richer. This finding suggests that sIVs have potential biological regulatory effects and may promote information exchange between different organelles in cells. By performing candidate gene enrichment analysis on differentially expressed miRNAs in sEVs and sIVs, we further found that sIVs are closely associated with intracellular membrane-like organelles. In summary, sEVs and sIVs differ in small RNA components, especially miRNAs. Enrichment analysis of miRNAs further confirmed that sIVs play an important role in intracellular material transport. Compared with sEVs, sIVs contain more diverse miRNAs and may have richer biological functions. These findings provide important clues for a deeper understanding of the function of sIVs in cell biology.
实施例7：sIVs具有独特的脂质组学特征Example 7: sIVs have unique lipidomic characteristics
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表8实验试剂Table 8 Experimental reagents
1.2实验仪器1.2 Experimental instruments
表9实验仪器Table 9 Experimental instruments
2、实验方法2. Experimental methods
2.1代谢物提取2.1 Metabolite extraction
分离富集sEVs和sIVs（实施例2制备）后使用PBS重悬。向容器中加入200μL的水，随后添加480μL的提取液，该提取液由MTBE和MeOH以5:1的比例混合而成，并含有内标物质。将混合液迅速放入液氮罐中冷冻1分钟，然后取出解冻，并通过涡旋混合器混合30秒，使溶液均匀。重复上述冷冻、解冻和混合步骤3次，接着在冰水浴中进行超声波处理10分钟。将处理后的样品在-40℃下静置1小时。然后，在4℃下以3000rpm（离心力900×g，半径8.6cm）离心15分钟，使样品分层。从上清液中取出300μL，转移至EP管中，并进行真空干燥。向干燥后的样品中加入100μL的复溶液（DCM:MeOH = 1:1），涡旋混合30秒，并在冰水浴中再次进行超声波处理10分钟。最后，在4℃下以13000rpm（离心力16200×g，半径8.6cm）离心15分钟，取75μL的上清液转移至进样瓶中，准备进行上机检测。After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS. 200 μL of water was added to the container, followed by 480 μL of an extract solution, which was a mixture of MTBE and MeOH in a ratio of 5:1 and contained an internal standard substance. The mixed solution was quickly placed in a liquid nitrogen tank and frozen for 1 minute, then taken out for thawing and mixed by a vortex mixer for 30 seconds to make the solution uniform. The above freezing, thawing and mixing steps were repeated 3 times, followed by ultrasonic treatment in an ice water bath for 10 minutes. The treated sample was allowed to stand at -40°C for 1 hour. Then, the sample was separated by centrifugation at 3000 rpm (centrifugal force 900 × g, radius 8.6 cm) at 4°C for 15 minutes. 300 μL was taken out of the supernatant, transferred to an EP tube, and vacuum dried. 100 μL of the complex solution (DCM: MeOH = 1:1) was added to the dried sample, vortexed for 30 seconds, and ultrasonically treated again in an ice water bath for 10 minutes. Finally, centrifuge at 13,000 rpm (centrifugal force 16,200 × g, radius 8.6 cm) for 15 min at 4 °C, and transfer 75 μL of the supernatant to a sample injection bottle for testing on the instrument.
2.2代谢物检测2.2 Metabolite detection
采用了Vanquish超高效液相色谱仪，并利用Waters ACQUITY UPLC HSS T3(2.1mm × 100mm, 1.8μm)液相色谱柱对目标化合物进行色谱分离。对于液相色谱，A相为含有10mmol/L甲酸铵的40%水溶液和60%乙腈溶液；B相为含有50mL/1000mL（10mmol/L）甲酸铵水溶液的10%乙腈和90%异丙醇溶液。我们采用了以下梯度洗脱程序：0~1.0min, 40% B；1.0~12.0min, 线性增加至100% B；12.0~13.5min, 保持100% B；13.5~13.7min, 线性减少至40% B；13.7~18.0min, 保持40% B。流动相流速设定为0.3mL/min，柱温为55℃，样品盘温度为4℃，进样体积为2μL（正离子和负离子模式）。Vanquish ultra-high performance liquid chromatograph was used, and the target compounds were chromatographically separated using a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) liquid chromatography column. For liquid chromatography, phase A was a 40% aqueous solution containing 10 mmol/L ammonium formate and a 60% acetonitrile solution; phase B was a 10% acetonitrile and 90% isopropanol solution containing 50 mL/1000 mL (10 mmol/L) ammonium formate aqueous solution. We used the following gradient elution program: 0-1.0 min, 40% B; 1.0-12.0 min, linear increase to 100% B; 12.0-13.5 min, maintain 100% B; 13.5-13.7 min, linear decrease to 40% B; 13.7-18.0 min, maintain 40% B. The mobile phase flow rate was set at 0.3 mL/min, the column temperature was 55 °C, the sample tray temperature was 4 °C, and the injection volume was 2 μL (positive and negative ion modes).
同时，使用Thermo Q Exactive HFX质谱仪，在Xcalibur控制软件（版本：4.0.27，Thermo）的控制下，进行一级和二级质谱数据采集。具体参数如下：Sheath gas flow rate设为10Arb，Capillary temperature设为350℃，Full ms resolution设为120000，MS/MSresolution设为7500，Collision energy在NCE模式下设为10/30/60，Spray Voltage设为4kV（正离子模式）或-3.8kV（负离子模式）。At the same time, the primary and secondary mass spectrometry data were collected using a Thermo Q Exactive HFX mass spectrometer under the control of Xcalibur control software (version: 4.0.27, Thermo). The specific parameters were as follows: Sheath gas flow rate was set to 10Arb, Capillary temperature was set to 350°C, Full ms resolution was set to 120000, MS/MSresolution was set to 7500, Collision energy was set to 10/30/60 in NCE mode, and Spray Voltage was set to 4 kV (positive ion mode) or -3.8 kV (negative ion mode).
2.3数据分析2.3 Data Analysis
使用ProteoWizard软件将质谱原始转成mzXML格式。再使用XCMS进行保留时间矫正、峰识别、峰提取、峰积分、峰对齐，minfrac设为0.5，cutoff设为0.3。使用XCMS软件、自撰写R程序包及lipidblast数据库进行脂质鉴定。生信绘图使用Hiplot在线绘制并使用AdobeIllustrator进行调整。ProteoWizard software was used to convert the mass spectra into mzXML format. XCMS was then used for retention time correction, peak identification, peak extraction, peak integration, and peak alignment, with minfrac set to 0.5 and cutoff set to 0.3. Lipid identification was performed using XCMS software, a self-written R package, and the lipidblast database. Bioinformatics graphics were drawn online using Hiplot and adjusted using Adobe Illustrator.
3、实验结果3. Experimental results
Orbitrap平台的电离源为电喷雾电离，有正离子模式（positive ion mode, POS）和负离子模式（negative ion mode, NEG）两种电离方式，在检测代谢组时结合使用两种方式可以使代谢物覆盖率更高，检测效果也更好，在数据分析时一般选取一种离子模式进行，本研究以正离子模式为例进行分析。The ionization source of the Orbitrap platform is electrospray ionization, which has two ionization modes: positive ion mode (POS) and negative ion mode (NEG). Combining the two modes when detecting metabolomes can achieve higher metabolite coverage and better detection effects. Generally, one ion mode is selected for data analysis. This study takes the positive ion mode as an example for analysis.
3.1 sEVs和sIVs中各类代谢物占比3.1 Proportion of various metabolites in sEVs and sIVs
脂质组学的二级谱图具有偶然性，因此在一组对比信息中所有组都鉴定到的脂质物才具有可信度。因此我们根据化学分类归属信息对不同细胞鉴定到的代谢物进行分类统计，各类代谢物占比如图32所示，MSCs鉴定到31种。sEVs和sIVs中多种脂质物表达水平存在差异。PC和PE是细胞膜常见的脂质成分，图32结果显示囊泡中PE和PC的占比均较高，这表明sEVs和sIVs的存在大量生物膜结构。The secondary spectra of lipidomics are accidental, so only lipids identified in all groups in a set of comparative information are credible. Therefore, we classified and counted the metabolites identified by different cells according to the chemical classification information. The proportion of each type of metabolite is shown in Figure 32. MSCs identified 31 types. There are differences in the expression levels of various lipids in sEVs and sIVs. PC and PE are common lipid components of cell membranes. The results in Figure 32 show that the proportion of PE and PC in vesicles is high, which indicates that sEVs and sIVs have a large number of biological membrane structures.
3.2 sEVs和sIVs中总脂质表达特征3.2 Total lipid expression characteristics in sEVs and sIVs
代谢组数据具有高通量的特点，采用主成分分析可以有效的突显代谢组学数据的总体分布趋势以及组间样本的差异程度。结果显示MSCs细胞sEVs和sIVs具有不同的脂质分布模式（图33），即sIVs是一种独特的囊泡群体，区别于sEVs，具有显著不同的脂质表达模式。Metabolomics data has the characteristics of high throughput, and principal component analysis can effectively highlight the overall distribution trend of metabolomics data and the degree of difference between samples in different groups. The results showed that MSCs cell sEVs and sIVs had different lipid distribution patterns (Figure 33), that is, sIVs is a unique vesicle population, different from sEVs, with significantly different lipid expression patterns.
3.3 sEVs和sIVs中差异脂质表达特征3.3 Differential lipid expression characteristics in sEVs and sIVs
热图可以直观展示组间代谢物差异的整体分布情况，我们将筛选差异代谢物的结果以热图的形式进行可视化。sIVs组对sEVs组的结果如图34所示。Heat maps can intuitively display the overall distribution of metabolite differences between groups. We visualized the results of screening differential metabolites in the form of heat maps. The results of the sIVs group versus the sEVs group are shown in Figure 34.
3.4 sEVs和sIVs中差异脂质的含量变化程度及分类信息3.4 Changes in the content and classification of differential lipids in sEVs and sIVs
脂质组柱形图利用代谢物的含量变化程度及分类信息来进行可视化的展示，sIVs组对sEVs组的结果如图35所示，脂质组柱形图中每个柱子代表一个代谢物。在MSCs中PC、PI、PE、PG和OxPI在sIVs中显著高表达，其中PC在sIVs中高表达200多倍。The lipidome bar graph uses the content change degree and classification information of metabolites for visualization. The results of the sIVs group versus the sEVs group are shown in Figure 35. Each column in the lipidome bar graph represents a metabolite. In MSCs, PC, PI, PE, PG, and OxPI are significantly overexpressed in sIVs, among which PC is overexpressed 200 times in sIVs.
4、小结4. Summary
脂质组学鉴定和量化各种脂质分子，脂质分为八大类，包括脂肪酰、甘油脂、磷脂、甾醇脂、丙烯醇脂、鞘脂、糖脂和聚酮。细胞膜主要含有各种磷脂，磷脂可以进一步分为甘油磷脂与鞘磷脂，二者具有明显的差异。甘油磷脂在细胞膜中主要位于磷脂双层的内小叶，与胆固醇一起构成细胞膜的主要成分，本实施例中sIVs含有较多的甘油磷脂，如PC和PE。鞘磷脂是一类含有鞘氨醇基团的磷脂，鞘磷脂位于细胞膜的外小叶，主要参与神经元的活动和信号传导，本实施例中sEVs含有较多的鞘磷脂，如SM。此外，甘油磷脂在生物体内还参与了许多其他生理过程，如能量代谢、激素合成等；而鞘磷脂在这些过程中的作用相对较小。sIVs中PC和PE表达水平较高，其中PC在sIVs中高表达200多倍。其中PC又被称为卵磷脂，被誉为与蛋白质、维生素并列的“第三营养素”，在生物学上具有多种重要作用。卵磷脂可以增加神经元的轴突生长、促进大脑发育，增强记忆力，并预防老年痴呆。此外，PE是构成生物膜骨架的主要分子之一。它的独特结构，包括一个磷酸基团、一个甘油、一个酰基和一种乙醇胺，使得它能够在生物膜中形成稳定的非片层以及多层脂质体泡囊。这种结构为生物膜提供了稳定的基础，有助于维持细胞的正常结构和功能。Lipidomics identifies and quantifies various lipid molecules. Lipids are divided into eight categories, including fatty acyl, glycerolipids, phospholipids, sterol lipids, propenol lipids, sphingolipids, glycolipids and polyketides. The cell membrane mainly contains various phospholipids, which can be further divided into glycerophospholipids and sphingomyelin, which have obvious differences. Glycerophospholipids are mainly located in the inner leaflet of the phospholipid bilayer in the cell membrane, and together with cholesterol, they constitute the main components of the cell membrane. In this example, sIVs contain more glycerophospholipids, such as PC and PE. Sphingomyelin is a type of phospholipid containing a sphingosine group. Sphingomyelin is located in the outer leaflet of the cell membrane and is mainly involved in neuronal activity and signal transduction. In this example, sEVs contain more sphingomyelin, such as SM. In addition, glycerophospholipids are also involved in many other physiological processes in the body, such as energy metabolism, hormone synthesis, etc.; while sphingomyelin plays a relatively small role in these processes. PC and PE expression levels are high in sIVs, among which PC is more than 200 times higher in sIVs. Among them, PC is also known as lecithin, which is known as the "third nutrient" alongside proteins and vitamins, and plays many important roles in biology. Lecithin can increase the axonal growth of neurons, promote brain development, enhance memory, and prevent Alzheimer's disease. In addition, PE is one of the main molecules that constitute the skeleton of biological membranes. Its unique structure, including a phosphate group, a glycerol, an acyl group, and an ethanolamine, enables it to form stable non-lamellar and multi-layer liposome vesicles in biological membranes. This structure provides a stable foundation for biological membranes and helps maintain the normal structure and function of cells.
有趣的是，sIVs高表达的PC和PE均属于甘油磷脂，内质网是甘油磷脂合成的场所，因此sIVs含有更多的甘油磷脂是合理的，进一步证实 sIVs是细胞内的组分，介导了细胞内的物质运输与各细胞器之间的交流。然而sEVs含有较多的鞘磷脂，sEVs源于细胞膜的内陷，并且经由细胞膜分泌到细胞外，因此含有较多的细胞膜外侧组分。这也表明sIVs缺乏外部膜结构，多种脂质的区别使sIVs与sEVs区分开来。先前的研究表明，蛋白质和脂质在细胞内的转运与膜弯曲和脂质分布效应有关。甘油磷脂可以通过调节链长和与胆固醇协同作用来调节膜的曲率和流动性，赋予sIVs更多的活力，从而持续参与细胞内的膜融合和裂变事件。Interestingly, the PC and PE highly expressed by sIVs are both glycerophospholipids. The endoplasmic reticulum is the site of glycerophospholipid synthesis, so it is reasonable that sIVs contain more glycerophospholipids, which further confirms that sIVs are intracellular components that mediate the transport of substances within the cell and the communication between organelles. However, sEVs contain more sphingomyelin. sEVs originate from the invagination of the cell membrane and are secreted to the outside of the cell through the cell membrane, so they contain more components outside the cell membrane. This also shows that sIVs lack an external membrane structure, and the difference in multiple lipids distinguishes sIVs from sEVs. Previous studies have shown that the transport of proteins and lipids in cells is related to membrane curvature and lipid distribution effects. Glycerophospholipids can regulate the curvature and fluidity of the membrane by adjusting the chain length and cooperating with cholesterol, giving sIVs more vitality, thereby continuously participating in membrane fusion and fission events within the cell.
在本实施例中，脂质组学数据进一步验证了sIVs区别于sEVs，为深入探索sIVs在细胞和组织相容性中的独特特性提供了重要的基础。In this example, lipidomics data further verified that sIVs are different from sEVs, providing an important basis for in-depth exploration of the unique properties of sIVs in cell and tissue compatibility.
实施例8：体外培养的细胞和体内的视网膜组织对sIVs具有更高的吸收效率Example 8: Cells cultured in vitro and retinal tissues in vivo have higher absorption efficiency for sIVs
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表10实验试剂Table 10 Experimental reagents
1.2实验仪器1.2 Experimental instruments
表11实验仪器Table 11 Experimental instruments
2、实验方法2. Experimental methods
2.1细胞培养2.1 Cell culture
人HRMECs购买自美国Angioproteomie公司。完整细胞培养基制备：向93ml ECM基本培养基中加入5ml的胎牛血清、1ml 生长因子和1ml青霉素-链霉素，得到含5%血清的完全培养基。RPE细胞完全培养基制备：向44.5ml DMEM基本培养基中加入5ml的胎牛血清、0.5ml青霉素-链霉素，得到含10%血清的完全培养基。使用低倍数显微镜查看细胞生长密度，采用高倍数显微镜观察细胞呈圆形、椭圆形或多边形扁平细胞，胞质中含有丰富成分，细胞内有很小的空泡。将细胞接种在培养瓶中，并在37℃的5% CO2培养箱中培养。培养基每3天更换一次。当细胞融合达到80%时，以1:2的继代比例进行传代，并使用P3至P5的细胞进行实验。Human HRMECs were purchased from Angioproteomie, USA. Preparation of complete cell culture medium: Add 5 ml of fetal bovine serum, 1 ml of growth factor and 1 ml of penicillin-streptomycin to 93 ml of ECM basic medium to obtain a complete medium containing 5% serum. Preparation of RPE cell complete culture medium: Add 5 ml of fetal bovine serum and 0.5 ml of penicillin-streptomycin to 44.5 ml of DMEM basic medium to obtain a complete medium containing 10% serum. Use a low-power microscope to check the cell growth density, and use a high-power microscope to observe that the cells are round, oval or polygonal flat cells, with rich components in the cytoplasm and small vacuoles in the cells. The cells were inoculated in a culture flask and cultured in a 5% CO2 incubator at 37 °C. The culture medium was changed every 3 days. When the cell fusion reached 80%, it was subcultured at a subculture ratio of 1:2, and cells from P3 to P5 were used for experiments.
2.2实验动物2.2 Experimental animals
健康雄性C57BL/6小鼠，8周龄，体重 21-22g，SPF级，购于北京维通利华公司（动物生产许可证：SCXK（京）2016-0006）。所有实验动物在室温下正常饮食，光照和黑暗时间分别12 h。动物饲养环境及实验操作内容均符合国家科学技术委员会有关《实验动物管理条例》的规定，并获得本院动物伦理委员会许可（伦理编号：TJYY2019091124）。将小鼠按照随机数字表法分组进行结膜下和视网膜下注射。Healthy male C57BL/6 mice, 8 weeks old, weighing 21-22 g, SPF grade, were purchased from Beijing Weitong Lihua Company (Animal Production License: SCXK (Beijing) 2016-0006). All experimental animals were fed a normal diet at room temperature, with a light and dark time of 12 h respectively. The animal breeding environment and experimental operations were in accordance with the provisions of the "Regulations on the Administration of Laboratory Animals" of the State Science and Technology Commission, and were approved by the Animal Ethics Committee of this hospital (Ethics Number: TJYY2019091124). The mice were divided into groups according to the random number table method for subconjunctival and subretinal injections.
2.3 DiD标记sIVs和sEVs与细胞共培养2.3 Co-culture of DiD-labeled sIVs and sEVs with cells
在37℃下囊泡与脂溶性示踪剂DiD溶液共孵育30分钟。通过Amicon® Ultra离心过滤器去除多余的DiD。将细胞接种到24孔培养板上预先放置的圆形片中，孵育24小时。将DiD标记的囊泡加入细胞的培养基中，于37℃孵育不同时间：3小时、12小时、24小时或48小时。除去培养基后，使用PBS清洗细胞，在室温下。使用4% PFA固定细胞10分钟，然后使用0.1% Triton X-100在室温下孵育5分钟以使细胞渗透。随后，细胞在室温下用CoraLite®Plus 488共轭的Phalloidin抗体染色30分钟。利用DAPI标记细胞核。最后，利用共聚焦激光扫描显微镜对细胞进行成像，通过Image J分析细胞内吞的囊泡量。Vesicles were incubated with a solution of lipid-soluble tracer DiD for 30 minutes at 37°C. Excess DiD was removed by Amicon® Ultra centrifugal filters. Cells were seeded into pre-placed circular pieces in 24-well culture plates and incubated for 24 hours. DiD-labeled vesicles were added to the culture medium of the cells and incubated at 37°C for different times: 3 hours, 12 hours, 24 hours, or 48 hours. After removing the culture medium, the cells were washed with PBS at room temperature. Cells were fixed with 4% PFA for 10 minutes and then incubated with 0.1% Triton X-100 for 5 minutes at room temperature to permeabilize the cells. Subsequently, cells were stained with CoraLite® Plus 488-conjugated Phalloidin antibody for 30 minutes at room temperature. Cell nuclei were labeled with DAPI. Finally, cells were imaged using confocal laser scanning microscopy and the amount of endocytosed vesicles was analyzed by Image J.
2.4结膜下注射和玻璃体腔注射DiD标记的sIVs和sEVs2.4 Subconjunctival and intravitreal injection of DiD-labeled sIVs and sEVs
如前述，囊泡用DiD染色。将DiD标记的囊泡以5μL的体积和3μg的质量注射到小鼠的结膜下。结膜下注射后24小时和48小时，收集眼球进行观察。将DiD标记的囊泡以1μL的体积和1μg的质量注入小鼠的玻璃体腔。玻璃体注射后8小时和48小时，收集眼球进行评估。Vesicles were stained with DiD as described above. DiD-labeled vesicles were injected into the subconjunctiva of mice at a volume of 5 μL and a mass of 3 μg. Eyeballs were collected for observation 24 hours and 48 hours after subconjunctival injection. DiD-labeled vesicles were injected into the vitreous cavity of mice at a volume of 1 μL and a mass of 1 μg. Eyeballs were collected for evaluation 8 hours and 48 hours after intravitreal injection.
2.5视网膜切片2.5 Retinal Sections
冷冻视网膜切片（8μm厚）用于免疫荧光分析。使用PFA在常温下对视网膜切片进行固定15分钟，随后用PBS洗涤载玻片，并使用DAPI对细胞核进行染色。最后，用抗荧光衰减封片剂封片，并使用共聚焦激光扫描显微镜观察。Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. Retinal sections were fixed with PFA for 15 minutes at room temperature, then the slides were washed with PBS and DAPI was used to stain the nuclei. Finally, the slides were mounted with anti-fluorescence attenuation mounting medium and observed using a confocal laser scanning microscope.
3、统计学处理3. Statistical processing
实验数据以均数±标准差（）表示。实验数据均进行正态检验。使用SPSS22.0分析所有定量数据。方差分析采取One-way ANOVA进行，使用最小显着性差异（LSD）分析进行事后检验。对于非正态分布数据和方差不均的数据，使用了非参数检验，P值<0.05为统计学显著差异。The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. All quantitative data were analyzed using SPSS22.0. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, nonparametric tests were used, andP values < 0.05 were considered statistically significant.
4、实验结果4. Experimental results
4.1细胞内吞sIVs能力优于sEVs4.1 Cells have a better ability to internalize sIVs than sEVs
为了评估细胞对两种类型囊泡的吞噬能力，选取RPE细胞和HRMECs与这MSC-sEVs（本实施例简称sEVs）和MSC-sIVs（本实施例简称sIVs）共同培养。简言之，将DiD标记的等量的囊泡与细胞一起孵育，在不同的时间点，使用共聚焦显微镜观察DiD的分布，以观察细胞对囊泡的摄取量。结果显示，在3小时的共培养后，细胞开始内化sEVs和sIVs，并且随着时间的推移，它们的摄取量继续增加（图36A和B，图37A和B）。在24小时时，sEVs和sIVs被细胞内化的量达到峰值，随后在24-48小时内逐渐减少（图36C，图37C）。值得注意的是，在RPE细胞和HRMECs中，在12至48小时期间，细胞对sIVs的内化率始终超过sEVs（图36C，图37C）。In order to evaluate the phagocytic ability of cells for the two types of vesicles, RPE cells and HRMECs were selected to co-culture with these MSC-sEVs (referred to as sEVs in this example) and MSC-sIVs (referred to as sIVs in this example). In brief, an equal amount of DiD-labeled vesicles were incubated with cells, and the distribution of DiD was observed using confocal microscopy at different time points to observe the cell uptake of vesicles. The results showed that after 3 hours of co-culture, cells began to internalize sEVs and sIVs, and their uptake continued to increase over time (Figures 36A and B, Figures 37A and B). At 24 hours, the amount of sEVs and sIVs internalized by cells reached a peak, and then gradually decreased within 24-48 hours (Figure 36C, Figure 37C). It is worth noting that in RPE cells and HRMECs, the internalization rate of sIVs by cells always exceeded sEVs from 12 to 48 hours (Figure 36C, Figure 37C).
4.2视网膜内吞sIVs能力优于sEVs4.2 The ability of retinal internalization of sIVs is superior to sEVs
为了评估视网膜对两种类型囊泡的吞噬能力，通过两种方法向眼睛施用等量的DiD标记的囊泡：结膜下注射和玻璃体内注射。在不同的时间点收集眼球样本，然后制备冷冻切片，并使用共聚焦显微镜观察视网膜内的囊泡摄取。结果显示，结膜下注射后24小时，sIVs穿透巩膜并进入视网膜下和视网膜间隙，而sEVs在结膜和巩膜之间积聚，到达视网膜下区域的sEVs较少（图38A）。48小时后，视网膜表现出对sEVs和sIVs的显著摄取（图38C）。具体而言，sEVs分布在RPE层，而sIVs广泛分布在外核层、内核层和神经节细胞层。玻璃体内注射DiD标记的囊泡后，囊泡从玻璃体腔向视网膜扩散。结果显示，注射后8小时，sEVs分布在玻璃体腔内，尚未到达视网膜，而sIVs已经到达整个视网膜层，并被神经节细胞层和内核层的细胞内化，在RPE层上观察到更高的sIVs积累（图38B）。玻璃体内注射24小时后，视网膜有效吸收sEVs和sIVs，两种类型的囊泡在整个视网膜中呈弥漫分布（图38B）。与sEVs相比，sIVs表现出更显著的摄取（图38D），并广泛分布于所有视网膜层，包括神经节细胞层、内丛状层、内核层、外丛状层、外核层和RPE层（图38B）。To evaluate the phagocytic capacity of the retina for both types of vesicles, an equal amount of DiD-labeled vesicles was administered to the eye by two methods: subconjunctival injection and intravitreal injection. Eyeball samples were collected at different time points, and then frozen sections were prepared and confocal microscopy was used to observe the vesicle uptake in the retina. The results showed that 24 hours after subconjunctival injection, sIVs penetrated the sclera and entered the subretinal and retinal spaces, while sEVs accumulated between the conjunctiva and sclera, and fewer sEVs reached the subretinal area (Figure 38A). After 48 hours, the retina showed significant uptake of sEVs and sIVs (Figure 38C). Specifically, sEVs were distributed in the RPE layer, while sIVs were widely distributed in the outer nuclear layer, inner nuclear layer, and ganglion cell layer. After intravitreal injection of DiD-labeled vesicles, the vesicles spread from the vitreous cavity to the retina. The results showed that 8 hours after injection, sEVs were distributed in the vitreous cavity and had not yet reached the retina, while sIVs had reached the entire retinal layer and were internalized by cells in the ganglion cell layer and inner nuclear layer, and higher sIVs accumulation was observed on the RPE layer (Figure 38B). 24 hours after intravitreal injection, the retina effectively absorbed sEVs and sIVs, and both types of vesicles were diffusely distributed throughout the retina (Figure 38B). Compared with sEVs, sIVs showed more significant uptake (Figure 38D) and were widely distributed in all retinal layers, including the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and RPE layer (Figure 38B).
5、小结5. Summary
本实施例表明体外培养的细胞对sIVs的内吞能力优于sEVs。通过对小鼠进行结膜下注射和玻璃体腔注射观察到视网膜对sIVs的内吞能力优于sEVs，这一发现充分展示了sIVs具有良好组织相容性。This example shows that cells cultured in vitro have better endocytosis of sIVs than sEVs. Subconjunctival injection and intravitreal injection in mice showed that the retina had better endocytosis of sIVs than sEVs, which fully demonstrated that sIVs have good tissue compatibility.
实施例9：MSC-sIVs对蓝光诱导的视网膜光损伤的保护作用Example 9: Protective effect of MSC-sIVs on blue light-induced retinal photodamage
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表12实验试剂Table 12 Experimental reagents
1.2、实验仪器1.2 Experimental instruments
表13实验仪器Table 13 Experimental instruments
2、实验方法2. Experimental methods
2.1实验动物2.1 Experimental animals
周龄为6周的BALB/c小鼠购自SPF（北京）生物技术有限公司（中国北京）。小鼠经历16小时的黑暗适应期，然后暴露于2000 Lux的蓝光下1小时，然后再回到黑暗中16小时。在蓝光照射之前，将托吡卡胺（0.5%）局部应用于角膜以扩大瞳孔。一个盒子的顶部有一个蓝色的灯光，四面都有镜子，用来进行光感应，盒子的每个角落都能接受大约2000Lux的照明。在蓝光照射前4小时，玻璃体内注射不同浓度的MSC-sEVs或MSC-sIVs（实施例2制备）（低浓度2μg和高浓度4μg），体积为1μL。使用相同量的PBS作为阴性对照。无蓝光损伤但注射1μLPBS的小鼠作为正常组。BALB/c mice aged 6 weeks were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). The mice underwent a 16-hour dark adaptation period and were then exposed to 2000 Lux of blue light for 1 hour and then returned to darkness for another 16 hours. Before blue light irradiation, tropicamide (0.5%) was topically applied to the cornea to dilate the pupil. A box with a blue light on the top and mirrors on all four sides for light sensing was illuminated at approximately 2000 Lux in each corner of the box. MSC-sEVs or MSC-sIVs (prepared in Example 2) with different concentrations (low concentration 2 μg and high concentration 4 μg) were injected intravitreally 4 hours before blue light irradiation in a volume of 1 μL. The same amount of PBS was used as a negative control. Mice without blue light injury but injected with 1 μL of PBS were used as the normal group.
2.2光学相干断层扫描2.2 Optical coherence tomography
将小鼠麻醉并使其瞳孔扩张。使用Spectralis HRA+OCT设备进行光谱域OCT，以检查视网膜结构变化。使用以视神经头为中心的环形扫描模式（圆直径1、3、6 ETDRS）测量视网膜厚度。Mice were anesthetized and their pupils were dilated. Spectral domain OCT was performed using the Spectralis HRA+OCT device to examine retinal structural changes. Retinal thickness was measured using a circular scanning pattern (circle diameter 1, 3, 6 ETDRS) centered on the optic nerve head.
2.3视网膜电图2.3 Electroretinogram
BABL/c小鼠在玻璃体腔注射后7天，经历暗适应12小时，然后使用机器（PhoenixResearch Labs，Pleasanton，CA）在黑暗中进行ERG评估。使用腹腔注射麻醉小鼠。麻醉后，金属电极位于角膜上，而参考电极位于前额和尾巴底部。ERG测量是利用由一系列闪光强度[0.1、1和2.2Log（cd.s/m2）]组成的光刺激获得的，每次闪光持续1ms。使用闪光诱导光感受器细胞（a波）和双极细胞反应（b波）。每次闪光后都会记录下来；三种反应的平均刺激间隔为1s（对于0.1Log（cd.s/m2））、10s（对于1Log（cd.sm2））或30s（对于2.2Log（cd.s/m2）），并记录振幅（μV）。BABL/c mice were dark-adapted for 12 h 7 days after intravitreal injection and then ERG assessments were performed in darkness using a machine (PhoenixResearch Labs, Pleasanton, CA). Mice were anesthetized using intraperitoneal injection. After anesthesia, metal electrodes were placed on the cornea, while reference electrodes were placed on the forehead and the base of the tail. ERG measurements were obtained using light stimulation consisting of a series of flash intensities [0.1, 1, and 2.2 Log (cd.s/m2)], each lasting 1 ms. Flashes were used to induce photoreceptor cell (a-wave) and bipolar cell responses (b-wave). Recordings were made after each flash; the three responses were averaged with an interstimulus interval of 1 s (for 0.1 Log (cd.s/m2)), 10 s (for 1 Log (cd.s/m2)), or 30 s (for 2.2 Log (cd.s/m2)), and the amplitude (μV) was recorded.
2.4小鼠视网膜的组织学评价2.4 Histological evaluation of mouse retina
BABL/c小鼠在玻璃体腔注射后7天，脱颈处死并收集眼球。将收集的眼球固定在4%多聚甲醛中并包埋在石蜡中。随后，从石蜡包埋的样品中制备4μm的视网膜水平切片，并用苏木精和伊红染色。使用显微镜（Zeiss，德国）捕获单个眼睛的每个切片的图像。BABL/c mice were killed by cervical dislocation 7 days after intravitreal injection and the eyeballs were collected. The collected eyeballs were fixed in 4% paraformaldehyde and embedded in paraffin. Subsequently, 4 μm horizontal sections of the retina were prepared from the paraffin-embedded samples and stained with hematoxylin and eosin. Images of each section of a single eye were captured using a microscope (Zeiss, Germany).
2.5免疫荧光染色2.5 Immunofluorescence staining
BABL/c小鼠在玻璃体腔注射后7天，脱颈处死并收集眼球。冷冻视网膜切片（8μm厚）用于免疫荧光分析。使用PFA在常温下进行固定15分钟，随后用PBS洗涤载玻片。载玻片在封闭缓冲液（10%正常山羊血清）中在室温下孵育1小时。将一抗应用于载玻片，并在4℃下孵育过夜。随后用PBS洗涤载玻片，并与与Alexa Fluor 488（稀释度1:5000）缀合的山羊抗兔IgG二级抗体在室温下孵育2小时。孵育后，用PBS冲洗载玻片，并使用DAPI对细胞核进行染色。最后，用抗荧光衰减封片剂封片，并使用共聚焦显微镜观察。BABL/c mice were killed by cervical dislocation 7 days after intravitreal injection and the eyeballs were collected. Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. PFA was used for fixation at room temperature for 15 minutes, and then the slides were washed with PBS. The slides were incubated in blocking buffer (10% normal goat serum) for 1 hour at room temperature. The primary antibody was applied to the slides and incubated overnight at 4°C. The slides were then washed with PBS and incubated with goat anti-rabbit IgG secondary antibody conjugated with Alexa Fluor 488 (dilution 1:5000) for 2 hours at room temperature. After incubation, the slides were rinsed with PBS and DAPI was used to stain the nuclei. Finally, the slides were mounted with anti-fluorescence attenuation mounting medium and observed using a confocal microscope.
2.6视网膜神经节细胞和小胶质细胞的定量2.6 Quantification of retinal ganglion cells and microglia
BABL/c小鼠在玻璃体腔注射后7天，脱颈处死并收集眼球。取出眼球并在4%多聚甲醛中固定30分钟。仔细解剖视网膜，用PBS冲洗，然后用含有1% Triton X-100的PBS透化30分钟。然后，用含有2% BSA和0.3% Triton X-100的PBS溶液在4℃下封闭视网膜2小时。然后用Rbpms和Iba1抗体对平板安装的视网膜进行染色，并在4℃的黑暗中孵育过夜，以观察视网膜神经节细胞和小胶质细胞。使用共聚焦荧光显微镜观察视网膜。如文献所述（Gharagozloo M., Smith M. D., Jin J., et al. Complement component 3 fromastrocytes mediates retinal ganglion cell loss during neuroinflammation [J].Acta Neuropathol, 2021, 142(5): 899-915），将视网膜分区域。使用ImageJ软件对视网膜神经节细胞和小胶质细胞进行定量。BABL/c mice were killed by cervical dislocation and eyeballs were collected 7 days after intravitreal injection. Eyeballs were removed and fixed in 4% paraformaldehyde for 30 minutes. The retinas were carefully dissected, rinsed with PBS, and then permeabilized with PBS containing 1% Triton X-100 for 30 minutes. Then, the retinas were blocked with PBS solution containing 2% BSA and 0.3% Triton X-100 at 4°C for 2 hours. Flat-mounted retinas were then stained with Rbpms and Iba1 antibodies and incubated overnight in the dark at 4°C to observe retinal ganglion cells and microglia. Retinas were observed using confocal fluorescence microscopy. The retinas were divided into regions as described in the literature (Gharagozloo M., Smith M. D., Jin J., et al. Complement component 3 fromastrocytes mediates retinal ganglion cell loss during neuroinflammation [J]. Acta Neuropathol, 2021, 142(5): 899-915). ImageJ software was used to quantify the number of retinal ganglion cells and microglia.
3、统计学处理3. Statistical processing
实验数据以均数±标准差（）表示。 实验数据均进行正态检验。使用SPSS22.0分析所有定量数据。方差分析采取One-way ANOVA进行，使用最小显着性差异（LSD）分析进行事后检验。对于非正态分布数据和方差不均的数据，使用了非参数检验，P值<0.05为统计学显著差异。The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. All quantitative data were analyzed using SPSS22.0. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, nonparametric tests were used, andP values < 0.05 were considered statistically significant.
4、实验结果4. Experimental results
4.1 MSC-sIVs保护视网膜结构免受蓝光损伤MSC-sIVs protect retinal structure from blue light damage
蓝光照射会导致视网膜结构损伤，主要表现为视网膜变薄。使用MSC-sEVs和MSC-sIVs对蓝光诱导的视网膜损伤进行治疗，并使用OCT和ERG评估视网膜结构和功能的变化。结果表明，蓝光照射后视网膜厚度显著减少（图39）。低剂量的MSC-sEVs和MSC-sIVs对视网膜变薄没有表现出显著的治疗效果；然而，高剂量的MSC-sEVs和MSC-sIVs显著改善了视网膜的变薄（图39C）。Blue light exposure can cause structural damage to the retina, mainly manifested as retinal thinning. MSC-sEVs and MSC-sIVs were used to treat blue light-induced retinal damage, and OCT and ERG were used to evaluate changes in retinal structure and function. The results showed that retinal thickness was significantly reduced after blue light exposure (Figure 39). Low doses of MSC-sEVs and MSC-sIVs did not show significant therapeutic effects on retinal thinning; however, high doses of MSC-sEVs and MSC-sIVs significantly improved retinal thinning (Figure 39C).
蓝光照射诱导视网膜损伤小鼠（本实施例简称BL）在7天后收集眼球，H&E染色显示，与未诱导的小鼠相比，用PBS治疗的BL小鼠的外核层（Outer Nuclear Layer, ONL）显著变薄，细胞出现大量丢失（图40A），光感受器细胞的内部和外部片段以及ONL层中的大多数细胞核丢失（图40A）。用MSC-sIVs治疗的BL小鼠这种变性情况大大减少（图40A）。视网膜切片中ONL细胞核计数的量化证实了PBS处理的小鼠因变性而导致的光感受器细胞广泛丢失，并证实了高剂量sEVs处理的BL小鼠以及低剂量和高剂量sIVs处理的BL小鼠视网膜损伤减轻（图40B）。Eyeballs of mice with retinal damage induced by blue light irradiation (abbreviated as BL in this example) were collected 7 days later, and H&E staining showed that the outer nuclear layer (ONL) of BL mice treated with PBS was significantly thinned, and a large number of cells were lost (Figure 40A), and the inner and outer fragments of photoreceptor cells and most of the nuclei in the ONL layer were lost (Figure 40A). This degeneration was greatly reduced in BL mice treated with MSC-sIVs (Figure 40A). Quantification of ONL nucleus counts in retinal sections confirmed the extensive loss of photoreceptor cells due to degeneration in PBS-treated mice, and confirmed that retinal damage was alleviated in BL mice treated with high-dose sEVs and low- and high-dose sIVs (Figure 40B).
4.2 MSC-sIVs保护视网膜电生理功能免受蓝光损伤MSC-sIVs protect retinal electrophysiological function from blue light damage
为了进一步研究高剂量MSC-sEVs和MSC-sIVs在改善视网膜电生理功能中的作用，我们使用ERG检测小鼠视网膜的功能。使用PBS治疗的BL小鼠的ERG a波和b波平均振幅在蓝光暴露七天后显著降低。图41A显示了每个治疗组中一个视网膜的代表性ERG波形。在高强度的光刺激下，用PBS处理的BL小鼠基本上失去了光感受器反应，波形呈熄灭状。在MSC-sEVs治疗组中，视网膜部分恢复了对光反应，而MSC-sIVs治疗组表现出显著改善的视网膜对光反应（图41B）。To further investigate the role of high-dose MSC-sEVs and MSC-sIVs in improving retinal electrophysiological function, we used ERG to detect mouse retinal function. The average amplitudes of the ERG a-wave and b-wave of BL mice treated with PBS were significantly reduced after seven days of blue light exposure. Figure 41A shows a representative ERG waveform of one retina in each treatment group. Under high-intensity light stimulation, BL mice treated with PBS essentially lost the photoreceptor response, and the waveform was extinguished. In the MSC-sEVs-treated group, the retina partially recovered its response to light, while the MSC-sIVs-treated group showed significantly improved retinal response to light (Figure 41B).
4.3 MSC-sIVs减少蓝光诱导的视网膜凋亡及光感受器细胞损伤4.3 MSC-sIVs reduce blue light-induced retinal apoptosis and photoreceptor cell damage
在蓝光诱导七天后收集来自处死小鼠的视网膜切片，并用TUNEL和DAPI染色以标记具有双链DNA断裂的细胞核（程序性细胞死亡或其他形式的细胞死亡的标志物）。代表性图像显示，凋亡信号主要集中在视网膜的外核层（图42A）。PBS处理的BL小鼠的视网膜显示出高水平的凋亡信号，而用MSC-sEVs和MSC-sIVs处理的BL小鼠显示出减少的凋亡信号。高剂量MSC-sIVs组显示出最显著的凋亡减少（图42B）。Retinal sections from sacrificed mice were collected seven days after blue light induction and stained with TUNEL and DAPI to mark nuclei with double-stranded DNA breaks (markers of programmed cell death or other forms of cell death). Representative images showed that apoptotic signals were mainly concentrated in the outer nuclear layer of the retina (Figure 42A). The retinas of PBS-treated BL mice showed high levels of apoptotic signals, while BL mice treated with MSC-sEVs and MSC-sIVs showed reduced apoptotic signals. The high-dose MSC-sIVs group showed the most significant reduction in apoptosis (Figure 42B).
为了进一步探索视网膜中感光细胞和突触连接的分布，用感光细胞的特异性标记物Rhodopsin（视紫红质）对视网膜切片进行染色。如代表性图像所示（图43A），视紫红质蛋白主要存在于视网膜的杆状光感受器细胞内，特别是这些细胞的外层，在光转导过程中发挥着至关重要的作用，即将光转化为电信号。在PBS处理的BL小鼠中，视紫红质蛋白的分布仅限于视网膜内少数感光细胞的外层，而用MSC-sEVs和MSC-sIVs处理的BL小鼠表现出更大的视紫红质蛋白阳性区域（图43B）。其中，与同等剂量的MSC-sEVs相比，高剂量的MSC-sIVs治疗显示出最高程度的预防视紫红质损失的作用（图43B）。对视网膜总蛋白提取物进行的Western Blot分析进一步支持了上述荧光定量结果（图44）。这些发现表明，与MSC-sEVs治疗相比，MSC-sIVs治疗可以更有效地减少光感受器细胞凋亡并改善突触连接。To further explore the distribution of photoreceptor cells and synaptic connections in the retina, retinal sections were stained with Rhodopsin, a specific marker for photoreceptor cells. As shown in representative images (Figure 43A), rhodopsin protein is mainly present in the rod photoreceptor cells of the retina, especially in the outer layer of these cells, and plays a vital role in phototransduction, which converts light into electrical signals. In PBS-treated BL mice, the distribution of rhodopsin protein was limited to the outer layer of a few photoreceptor cells in the retina, while BL mice treated with MSC-sEVs and MSC-sIVs showed larger rhodopsin protein-positive areas (Figure 43B). Among them, high-dose MSC-sIVs treatment showed the highest degree of prevention of rhodopsin loss compared with the same dose of MSC-sEVs (Figure 43B). Western Blot analysis of retinal total protein extracts further supported the above fluorescence quantification results (Figure 44). These findings suggest that MSC-sIVs treatment can more effectively reduce photoreceptor cell apoptosis and improve synaptic connections compared with MSC-sEVs treatment.
4.4 MSC-sIVs减少蓝光诱导的视网膜炎症激活MSC-sIVs reduce blue light-induced retinal inflammatory activation
暴露在强烈的蓝光下会导致氧化应激和炎症，从而导致视网膜细胞死亡和随后的视力损伤。GFAP是活化的Müller细胞的标志物，表明视网膜存在炎症。在蓝光暴露后七天被安乐死的小鼠的眼部切片上进行GFAP的免疫荧光染色。在正常小鼠的眼部切片中，GFAP染色主要局限于内界膜附近，外丛状层的染色较少（图45A）。在暴露于蓝光并用PBS处理的小鼠的眼睛中，观察到跨越内部丛状层的显著GFAP染色（图45A）。这种额外的信号在暴露于蓝光但用MSC-sEVs和MSC-sIVs处理的小鼠中减弱（图45A）。对GFAP信号的定量证实了BL小鼠视网膜中GFAP表达的统计学显著增加，但低剂量和高剂量MSC-sEVs和MSC-sIVs的治疗都阻止了这种增加，高剂量MSC-sIVs治疗产生了最显著的效果（图45A和图45B）。对总视网膜蛋白提取物进行的蛋白质印迹分析进一步支持了上述荧光定量结果（图44）。Exposure to intense blue light causes oxidative stress and inflammation, which leads to retinal cell death and subsequent visual impairment. GFAP is a marker of activated Müller cells, indicating the presence of inflammation in the retina. Immunofluorescence staining for GFAP was performed on ocular sections of mice euthanized seven days after blue light exposure. In ocular sections of normal mice, GFAP staining was mainly confined to the vicinity of the inner limiting membrane, with less staining in the outer plexiform layer (Figure 45A). In the eyes of mice exposed to blue light and treated with PBS, significant GFAP staining across the inner plexiform layer was observed (Figure 45A). This additional signal was attenuated in mice exposed to blue light but treated with MSC-sEVs and MSC-sIVs (Figure 45A). Quantification of the GFAP signal confirmed a statistically significant increase in GFAP expression in the retina of BL mice, but treatment with both low and high doses of MSC-sEVs and MSC-sIVs prevented this increase, with high-dose MSC-sIVs treatment producing the most significant effect (Figures 45A and 45B). Western blot analysis of total retinal protein extracts further supported the above fluorescence quantification results (Figure 44).
为了进一步评估MSC-sIVs治疗对BL小鼠视网膜炎症的影响，使用Iba1标记小鼠视网膜铺片中的小胶质细胞，并在视网膜内层和外层进行细胞计数。在未暴露于蓝光的小鼠中，在视网膜内层检测到排列整齐；形状规则的Iba1阳性细胞，而在视网膜外层发现少量的Iba1阳性细胞（图46A）。但是在接受蓝光照射的小鼠视网膜发现大量的Iba1阳性细胞，呈不规则排列，在MSC-sEVs和MSC-sIVs治疗组中，这种增加受到不同程度的抑制。细胞计数显示，与sEVs相比，相同剂量的sIVs具有更显著的抑制作用（图46B、图46C和图46D）。这些数据表明，蓝光暴露导致视网膜外层出现小胶质细胞阳性细胞，MSC-sIVs在很大程度上抑制了小胶质细胞增多。To further evaluate the effect of MSC-sIVs treatment on retinal inflammation in BL mice, Iba1 was used to label microglia in mouse retinal flat mounts, and cell counts were performed in the inner and outer layers of the retina. In mice not exposed to blue light, neatly arranged and regularly shaped Iba1-positive cells were detected in the inner layer of the retina, while a small number of Iba1-positive cells were found in the outer layer of the retina (Figure 46A). However, a large number of Iba1-positive cells were found in the retina of mice irradiated with blue light, which were arranged irregularly, and this increase was inhibited to varying degrees in the MSC-sEVs and MSC-sIVs treatment groups. Cell counting showed that sIVs at the same dose had a more significant inhibitory effect compared with sEVs (Figure 46B, Figure 46C, and Figure 46D). These data indicate that blue light exposure leads to the appearance of microglia-positive cells in the outer layer of the retina, and MSC-sIVs largely inhibit the increase in microglia.
5、小结5. Summary
在本实施例中，我们使用MSC-sEVs和MSC-sIVs同时对蓝光损伤视网膜病变模型小鼠进行干预。结果显示MSC-sIVs可以减缓视网膜变薄，在高剂量时效果更佳，其中MSC-sIVs在减缓细胞核丢失和电生理功能改善方面优于MSC-sEVs。此外，高剂量MSC-sIVs在减少视网膜炎症和凋亡方面更显著。我们的研究证明MSC-sIVs在视网膜光损伤小鼠模型中的保护作用。In this example, we used MSC-sEVs and MSC-sIVs to intervene in the blue light-damaged retinopathy model mice at the same time. The results showed that MSC-sIVs can slow down retinal thinning, and the effect is better at high doses, among which MSC-sIVs are superior to MSC-sEVs in slowing down cell nuclear loss and improving electrophysiological function. In addition, high-dose MSC-sIVs are more significant in reducing retinal inflammation and apoptosis. Our study demonstrates the protective effect of MSC-sIVs in the retinal light damage mouse model.
实施例10：MSC-sIVs对rd10小鼠视网膜变性的保护作用Example 10: Protective effect of MSC-sIVs on retinal degeneration in rd10 mice
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表14实验试剂Table 14 Experimental reagents
1.2实验仪器1.2 Experimental instruments
表15实验仪器Table 15 Experimental instruments
2、实验方法2. Experimental methods
2.1实验动物2.1 Experimental animals
rd10小鼠由西南医科大学徐海伟教授提供，C57BL/6J（野生型，WT）对照小鼠购自SPF（北京）生物技术有限公司（中国北京）。14天的rd10小鼠用MSC-sEVs、MSC-sIVs或PBS处理（对照组）。动物被安置在实验室动物部的标准、无特定病原体（SPF）的条件下，具有12小时的光/暗周期，每个笼子4-5只小鼠。本研究中的小鼠维持在标准的正常饮食中。所有涉及动物的手术均按照ARVO关于动物在眼科和视觉研究中的使用说明和天津医科大学眼科研究所动物研究指南进行。rd10 mice were provided by Professor Xu Haiwei of Southwest Medical University, and C57BL/6J (wild type, WT) control mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). 14-day-old rd10 mice were treated with MSC-sEVs, MSC-sIVs, or PBS (control group). Animals were housed under standard, specific pathogen-free (SPF) conditions in the laboratory animal department with a 12-h light/dark cycle, with 4–5 mice per cage. Mice in this study were maintained on a standard normal diet. All surgeries involving animals were performed in accordance with the ARVO instructions for the use of animals in ophthalmic and visual research and the guidelines for animal research of the Institute of Ophthalmology, Tianjin Medical University.
2.2小鼠玻璃体腔注射2.2 Intravitreal injection in mice
在rd10小鼠发育至p12时，进行玻璃体腔注射。使用复方托品卡胺散瞳，使用盐酸利多卡因麻醉眼表，使用33G微量注射器进行玻璃体腔注射。MSC-sEVs和MSC-sIVs（实施例2制备）体积为1μL，质量为2μg。同样体积的PBS作为对照。When rd10 mice developed to p12, intravitreal injection was performed. Tropicamide compound was used to dilate the pupil, lidocaine hydrochloride was used to anesthetize the ocular surface, and a 33G microsyringe was used for intravitreal injection. The volume of MSC-sEVs and MSC-sIVs (prepared in Example 2) was 1 μL and the mass was 2 μg. The same volume of PBS was used as a control.
2.3视网膜电图2.3 Electroretinogram
同实施例9的2.3。Same as 2.3 of Example 9.
2.4小鼠视网膜的组织学评价2.4 Histological evaluation of mouse retina
同实施例9的2.4。Same as 2.4 of Example 9.
2.5免疫荧光染色2.5 Immunofluorescence staining
同实施例9的2.5。Same as 2.5 of Example 9.
2.6视网膜小胶质细胞的定量2.6 Quantification of retinal microglia
同实施例9的2.6。Same as 2.6 of Example 9.
3、统计学处理3. Statistical processing
同实施例9的3。Same as 3 of Example 9.
4、实验结果4. Experimental results
4.1 MSC-sIVs对rd10小鼠视网膜外核层细胞的保护作用4.1 Protective effect of MSC-sIVs on retinal outer nuclear layer cells of rd10 mice
为了评估MSC-sIVs的保护作用，我们最初使用H&E染色来观察p28视网膜ONL的结构和细胞数量。在p28时rd10小鼠的ONL细胞几乎完全丧失（图47A）。然而，MSC-sEVs和MSC-sIVs治疗都能够逆转这些变化，与MSC-sEVs相比，MSC-sIVs表现出明显显著的治疗效果（图47B）。To evaluate the protective effects of MSC-sIVs, we initially used H&E staining to observe the structure and cell number of the ONL in the retina at p28. There was an almost complete loss of ONL cells in rd10 mice at p28 (Figure 47A). However, both MSC-sEVs and MSC-sIVs treatment were able to reverse these changes, with MSC-sIVs showing a significantly more significant therapeutic effect compared to MSC-sEVs (Figure 47B).
4.2 MSC-sIVs对rd10小鼠视网膜功能的保护作用4.2 Protective effect of MSC-sIVs on retinal function in rd10 mice
我们使用ERG评估rd10小鼠在p28的视网膜电生理功能。在p28时，未经治疗的rd10小鼠的ERG波形表现出熄灭状态，代表光感受器细胞功能的全面崩溃（图48A）。在所有测试的闪光强度下，rd10小鼠的光反应显著减少（图48B），MSC-sEVs和MSC-sIVs治疗显著挽救了rd10视网膜的光反应（图48B）。从统计数据来看，MSC-sEVs在中等至高刺激（>1.0 cd•s/m2）下显著提高了a波和b波振幅，而MSC-sIVs在低、中等和高刺激（>0.1 cd•s/m2）下显著增强了a波或b波振幅。此外，MSC-sIVs在最高刺激下的a波振幅和在低、中、高刺激（>0.1 cd•s/m2）下的b波振幅的改善显著优于MSC-sEVs。总之，我们的研究结果表明MSC-sIVs增强了rd10小鼠的视觉功能。We used ERG to evaluate the electrophysiological function of the retina of rd10 mice at p28. At p28, the ERG waveform of untreated rd10 mice exhibited a quenched state, representing a comprehensive collapse of photoreceptor cell function (Figure 48A). The light response of rd10 mice was significantly reduced at all flash intensities tested (Figure 48B), and treatment with MSC-sEVs and MSC-sIVs significantly rescued the light response of rd10 retinas (Figure 48B). Statistically speaking, MSC-sEVs significantly increased a-wave and b-wave amplitudes at moderate to high stimulation (>1.0 cd•s/m2), while MSC-sIVs significantly enhanced a-wave or b-wave amplitudes at low, moderate, and high stimulation (>0.1 cd•s/m2). In addition, MSC-sIVs significantly outperformed MSC-sEVs in improving a-wave amplitude at the highest stimulation and b-wave amplitude at low, moderate, and high stimulation (>0.1 cd•s/m2). In conclusion, our results indicate that MSC-sIVs enhance visual function in rd10 mice.
4.3 MSC-sIVs对rd10小鼠视网膜凋亡的抑制作用4.3 Inhibitory effect of MSC-sIVs on retinal apoptosis in rd10 mice
在p28，收集rd10小鼠的视网膜切片，并用TUNEL和DAPI染色，以用双链DNA断裂标记细胞核。代表性图像显示，凋亡信号主要集中在视网膜的外核层（图49A）。相反，用MSC-sEVs和MSC-sIVs治疗的rd10小鼠显示出减少的凋亡信号，与MSC-sEVs治疗组相比，在MSC-sIVs处理组中观察到的凋亡减少最为显著（图49B）。At p28, retinal sections of rd10 mice were collected and stained with TUNEL and DAPI to mark nuclei with double-stranded DNA breaks. Representative images showed that apoptotic signals were mainly concentrated in the outer nuclear layer of the retina (Figure 49A). In contrast, rd10 mice treated with MSC-sEVs and MSC-sIVs showed reduced apoptotic signals, with the most significant reduction in apoptosis observed in the MSC-sIVs-treated group compared to the MSC-sEVs-treated group (Figure 49B).
4.4 MSC-sIVs对rd10小鼠视网膜光感受器细胞的保护作用4.4 Protective effect of MSC-sIVs on retinal photoreceptor cells in rd10 mice
视紫红质染色用于进一步检查视网膜的光感受器细胞。在rd10小鼠中，视网膜中光感受器细胞的缺失导致视紫红质蛋白表达减少（图50）。然而，与MSC-sEVs相比，MSC-sIVs表现出更显著的治疗效果（图50）。Rhodopsin staining was used to further examine the photoreceptor cells of the retina. In rd10 mice, the loss of photoreceptor cells in the retina resulted in reduced rhodopsin protein expression (Figure 50). However, MSC-sIVs showed a more significant therapeutic effect compared to MSC-sEVs (Figure 50).
同时，为了进一步研究MSC-sIVs对rd10小鼠视网膜突触连接的影响，使用PSD95对视网膜切片进行染色。PSD95是一种突触后相关蛋白，在视网膜中，位于感光神经元的突触前末端。在rd10视网膜中，观察到PSD95表达明显较弱的染色和条带结构的丧失（图51A）。然而，MSC-sEVs和MSC-sIVs治疗都能够挽救PSD95表达的损失，MSC-sIVs的恢复效果明显优于MSC-sEVs（图51B）。Meanwhile, to further investigate the effects of MSC-sIVs on synaptic connectivity in the retina of rd10 mice, retinal sections were stained with PSD95. PSD95 is a postsynaptic-associated protein that is located at the presynaptic terminals of photoreceptor neurons in the retina. In the rd10 retina, significantly weaker staining and loss of the band structure of PSD95 expression were observed (Figure 51A). However, both MSC-sEVs and MSC-sIVs treatment were able to rescue the loss of PSD95 expression, and the recovery effect of MSC-sIVs was significantly better than that of MSC-sEVs (Figure 51B).
4.5 MSC-sIVs对rd10小鼠炎症的抑制作用4.5 Inhibitory effect of MSC-sIVs on inflammation in rd10 mice
我们使用免疫染色评估了GFAP在WT和rd10小鼠视网膜中的表达。如前所述，GFAP主要在位于正常视网膜神经节细胞层的星形胶质细胞中表达。相反，在rd10组中，GFAP染色沿着整个视网膜的Müller神经胶质扩展（图52A）。MSC-sEVs和MSC-sIVs处理降低了Müller细胞中GFAP的表达。对GFAP荧光强度的量化表明，与MSC-sEVs治疗组相比，MSC-sIVs治疗组表现出明显显著的治疗效果（图52B）。使用Western Blot进一步证实上述荧光定量分析结果（图55）。We evaluated the expression of GFAP in the retinas of WT and rd10 mice using immunostaining. As mentioned above, GFAP is mainly expressed in astrocytes located in the ganglion cell layer of the normal retina. In contrast, in the rd10 group, GFAP staining extended along the Müller glia throughout the retina (Figure 52A). MSC-sEVs and MSC-sIVs treatment reduced the expression of GFAP in Müller cells. Quantification of GFAP fluorescence intensity showed that the MSC-sIVs treatment group showed a significantly significant therapeutic effect compared with the MSC-sEVs treatment group (Figure 52B). The above fluorescence quantitative analysis results were further confirmed using Western Blot (Figure 55).
通过Iba1染色检测小胶质细胞的活性状态。在rd10小鼠中，发现视网膜中的Iba1阳性细胞紊乱，视网膜内层和外层的数量增加（图53A）。在rd10视网膜的所有层中都观察到Iba1阳性细胞，特别是在ONL和外节段内，小胶质细胞表现出反应性形态（其特征是细胞体增大和突起缩回）（图53A，图54）。MSC-sEVs和MSC-sIVs处理显著减少了视网膜内层和视网膜外层中Iba1阳性细胞的数量。具体而言，MSC-sIVs在减少视网膜外层和整个视网膜中的Iba1阳性细胞数量方面优于sEVs（图53B）。The activity state of microglia was detected by Iba1 staining. In rd10 mice, Iba1-positive cells in the retina were found to be disorganized, and the number in the inner and outer layers of the retina increased (Figure 53A). Iba1-positive cells were observed in all layers of the rd10 retina, especially in the ONL and outer segments, and microglia exhibited a reactive morphology (characterized by enlarged cell bodies and retracted processes) (Figure 53A, Figure 54). MSC-sEVs and MSC-sIVs treatment significantly reduced the number of Iba1-positive cells in the inner and outer layers of the retina. Specifically, MSC-sIVs were superior to sEVs in reducing the number of Iba1-positive cells in the outer retina and the entire retina (Figure 53B).
5、小结5. Summary
本实施例使用MSC-sEVs和MSC-sIVs同时对rd10模型小鼠进行干预。与MSC-sEVs相比，MSC-sIVs可以更大程度减缓视网膜变性导致的视网膜光感受器细胞丢失。MSC-sIVs可以减少视网膜光感受器细胞凋亡，小胶质细胞激活。This example uses MSC-sEVs and MSC-sIVs to intervene in rd10 model mice at the same time. Compared with MSC-sEVs, MSC-sIVs can slow down the loss of retinal photoreceptor cells caused by retinal degeneration to a greater extent. MSC-sIVs can reduce retinal photoreceptor cell apoptosis and microglia activation.
实施例11：MSC-sIVs通过抑制内质网应激改善视网膜损伤Example 11: MSC-sIVs improve retinal damage by inhibiting endoplasmic reticulum stress
1、实验仪器和材料1. Experimental instruments and materials
1.1实验试剂1.1 Experimental Reagents
表16实验试剂Table 16 Experimental reagents
1.2实验仪器1.2 Experimental instruments
表17实验仪器Table 17 Experimental instruments
2、实验方法2. Experimental methods
2.1视网膜蛋白提取2.1 Retinal protein extraction
对于Babl/c小鼠，在蓝光照射后7天，脱颈处死小鼠，取眼球，取视网膜放入EP管中，液氮速冻后保存至-80℃。对于rd10小鼠，在p21和p28，脱颈处死小鼠，取眼球，取视网膜放入EP管中，液氮速冻后保存至-80℃。For Babl/c mice, 7 days after blue light exposure, the mice were killed by dislocating the neck, and the eyeballs were removed. The retinas were placed in EP tubes, frozen in liquid nitrogen, and stored at -80°C. For rd10 mice, the mice were killed by dislocating the neck at p21 and p28, and the eyeballs were removed. The retinas were placed in EP tubes, frozen in liquid nitrogen, and stored at -80°C.
向1个视网膜加入150μL含有蛋白酶抑制剂的Ripa蛋白裂解液，使用枪头吹打视网膜，并超声破碎视网膜。超声条件为：冰上，功率35%，ON 2s，OFF 2s，共计1分钟。随后在4℃条件下，12000g离心15分钟，取上清液为视网膜蛋白。Add 150 μL of Ripa protein lysis buffer containing protease inhibitors to one retina, blow the retina with a pipette tip, and ultrasonically break the retina. The ultrasonic conditions are: on ice, power 35%, ON 2s, OFF 2s, a total of 1 minute. Then centrifuge at 12000g for 15 minutes at 4°C, and take the supernatant as retinal protein.
2.2视网膜mRNA-seq2.2 Retinal mRNA-seq
2.2.1提取RNA及构建文库2.2.1 Extraction of RNA and construction of library
采用Agilent 2100 bioanalyzer评估RNA的完整性和总量，确保样本的高质量。使用total RNA构建文库。使用Oligo(dT)磁珠富集带有polyA尾的mRNA，随后在特定的Fragmentation Buffer中，使用二价阳离子将这些mRNA随机打断。以这些片段化的mRNA为模板，利用随机寡核苷酸作为引物，在M-MuLV逆转录酶体系中合成cDNA的第一条链。然后使用RNaseH来降解RNA链，并在DNA polymerase I体系下，以dNTPs为原料，合成cDNA的第二条链。经过纯化的双链cDNA会进行末端修复、加A尾，并与测序接头连接。利用AMPure XPbeads筛选出大约370~420 bp的cDNA片段，进行PCR扩增，并再次使用AMPure XP beads来纯化PCR产物，最终得到所需的文库。The integrity and total amount of RNA were assessed using an Agilent 2100 bioanalyzer to ensure high sample quality. Total RNA was used to construct the library. Oligo(dT) magnetic beads were used to enrich mRNA with polyA tails, and then these mRNAs were randomly sheared using divalent cations in a specific Fragmentation Buffer. These fragmented mRNAs were used as templates and random oligonucleotides were used as primers to synthesize the first strand of cDNA in the M-MuLV reverse transcriptase system. RNaseH was then used to degrade the RNA strand, and the second strand of cDNA was synthesized using dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tailed, and ligated to the sequencing adapter. cDNA fragments of approximately 370-420 bp were screened using AMPure XP beads, amplified by PCR, and the PCR products were purified again using AMPure XP beads to obtain the desired library.
完成文库构建后，首先使用Qubit2.0 Fluorometer进行初步定量，将文库稀释至1.5ng/ul。然后再用Agilent 2100 bioanalyzer来检测文库的insert size，确保其符合预期。最后，通过qRT-PCR来准确测定文库的有效浓度（确保有效浓度高于2nM），以保证文库的高质量。After the library construction is completed, the Qubit2.0 Fluorometer is used for preliminary quantification, and the library is diluted to 1.5ng/ul. Then the Agilent 2100 bioanalyzer is used to detect the insert size of the library to ensure that it meets expectations. Finally, qRT-PCR is used to accurately determine the effective concentration of the library (to ensure that the effective concentration is higher than 2nM) to ensure the high quality of the library.
2.2.2上机测序2.2.2 Sequencing
使用Illumina NovaSeq 6000进行测序，产生150 bp的配对末端读数。测序的基本原理是边合成边测序。在测序的flow cell中，我们加入四种荧光标记的dNTP、DNA聚合酶以及接头引物进行扩增。每当测序簇延伸互补链并加入一个被荧光标记的dNTP时，就会释放出相应的荧光。测序仪通过捕获这些荧光信号，并利用计算机软件将光信号转化为测序峰，从而获取待测片段的序列信息。Sequencing was performed using the Illumina NovaSeq 6000, generating 150 bp paired-end reads. The basic principle of sequencing is sequencing by synthesis. In the sequencing flow cell, we added four fluorescently labeled dNTPs, DNA polymerase, and adapter primers for amplification. Whenever the sequencing cluster extends the complementary chain and adds a fluorescently labeled dNTP, the corresponding fluorescence is released. The sequencer captures these fluorescent signals and uses computer software to convert the light signals into sequencing peaks to obtain the sequence information of the fragment to be tested.
2.2.3数据处理与解析2.2.3 Data processing and analysis
高通量测序仪生成的图像数据，经过CASAVA的碱基识别处理，被转换为序列数据（reads）。原始数据中会包含一些带有测序接头或质量不高的reads，这些都需要进行过滤，以确保数据分析的准确性和可靠性。过滤步骤主要包括：移除带有接头（adapter）的reads，剔除含有无法确定碱基信息（N）的reads，以及清除质量较低的reads（即Qphred值小于等于20的碱基占整个read长度50%以上的reads）。同时，计算过滤后数据（clean data）的Q20、Q30值和GC含量。所有后续分析均基于这些净化后的数据进行。直接从权威的基因组网站获取参考基因组和基因模型注释文件。使用HISAT2（v2.0.5）构建参考基因组的索引，并将经过净化的配对末端reads与参考基因组进行比对。选择HISAT2作为比对工具的原因是，它能够根据基因模型注释文件生成拼接连接的数据库，从而提供比其他非拼接比对工具更精确的比对效果。The image data generated by the high-throughput sequencer is converted into sequence data (reads) after base recognition processing by CASAVA. The raw data will contain some reads with sequencing adapters or low quality, which need to be filtered to ensure the accuracy and reliability of data analysis. The filtering steps mainly include: removing reads with adapters, removing reads with undetermined base information (N), and removing low-quality reads (i.e., reads with bases with Qphred values less than or equal to 20 accounting for more than 50% of the entire read length). At the same time, the Q20, Q30 values and GC content of the filtered data (clean data) are calculated. All subsequent analyses are based on these purified data. The reference genome and gene model annotation files are obtained directly from the authoritative genome website. HISAT2 (v2.0.5) is used to build an index of the reference genome, and the purified paired-end reads are aligned with the reference genome. The reason for choosing HISAT2 as the alignment tool is that it can generate a spliced database based on the gene model annotation file, thus providing a more accurate alignment than other non-spliced alignment tools.
2.2.4基因表达量的量化分析2.2.4 Quantitative analysis of gene expression
采用featureCounts（1.5.0-p3）来计算映射到每个基因的读数。随后，根据基因的长度计算每个基因的FPKM值，即每百万碱基对测序的转录本序列片段的每千碱基片段的预期数量。这种方法同时考虑了测序深度和基因长度对读数计数的影响，是目前评估基因表达水平最常用的方法之一。FeatureCounts (1.5.0-p3) was used to calculate the number of reads mapped to each gene. Subsequently, the FPKM value of each gene was calculated based on the length of the gene, that is, the expected number of fragments per kilobase of transcript sequence fragments per million base pairs sequenced. This method takes into account the effects of sequencing depth and gene length on read counts and is currently one of the most commonly used methods for evaluating gene expression levels.
2.2.5差异表达分析2.2.5 Differential expression analysis
使用DESeq2软件（1.20.0）进行差异表达分析。DESeq2基于负二项式分布模型来确定数字基因表达数据中的差异表达，并提供相应的统计程序。通过Benjamini和Hochberg的方法来调整P值（padj），以控制错误发现率。显著差异表达的阈值设定为padj<=0.05且|log2（foldchange）|>=1。Differential expression analysis was performed using DESeq2 software (1.20.0). DESeq2 determines differential expression in digital gene expression data based on a negative binomial distribution model and provides corresponding statistical procedures. P values (padj) were adjusted by the method of Benjamini and Hochberg to control the false discovery rate. The threshold for significant differential expression was set at padj <= 0.05 and |log2(foldchange)|>=1.
2.2.6差异基因的富集分析2.2.6 Enrichment analysis of differentially expressed genes
利用clusterProfiler（3.8.1）软件对差异表达基因进行GO富集分析，并修正基因长度偏差。考虑具有校正后P值小于0.05的GO term为显著差异表达基因显著富集。此外，还使用clusterProfiler分析KEGG通路、Reactome通路、DO通路以及DisGeNET通路中差异表达基因的统计富集情况。这些数据库资源有助于从分子水平了解生物系统的高级功能和效用，如细胞、生物体和生态系统等。KEGG、Reactome、DO和DisGeNET的显著性富集阈值均设定为校正后的P值小于0.05。ClusterProfiler (3.8.1) software was used to perform GO enrichment analysis on differentially expressed genes and correct for gene length deviation. GO terms with corrected P values less than 0.05 were considered significantly enriched for significantly differentially expressed genes. In addition, clusterProfiler was also used to analyze the statistical enrichment of differentially expressed genes in KEGG pathways, Reactome pathways, DO pathways, and DisGeNET pathways. These database resources help to understand the advanced functions and utilities of biological systems such as cells, organisms, and ecosystems at the molecular level. The significant enrichment thresholds for KEGG, Reactome, DO, and DisGeNET were all set to a corrected P value less than 0.05.
2.3 Western Blot2.3 Western Blot
同实施例4的2.4。Same as 2.4 of Example 4.
3、统计学处理3. Statistical processing
同实施例9的3。Same as 3 of Example 9.
4、实验结果4. Experimental results
4.1 RNA-seq结果显示MSC-sIVs对光损伤视网膜的治疗作用4.1 RNA-seq results show the therapeutic effect of MSC-sIVs on light-damaged retina
为了探究MSC-sIVs对光损伤小鼠视网膜的治疗作用，我们收集了经过MSC-sIVs治疗后7天的光损伤小鼠视网膜（来自实施例9），与未经治疗（注射PBS）的光损伤小鼠视网膜进行RNA测序，结果显示以下通路存在显著差异：叶酸介导的一碳单位代谢池、钙信号通路、血管平滑肌收缩、cGMP-PKG信号通路、硫胺素代谢、辅因子生物合成、心肌收缩和催产素信号通路等（图56）。In order to explore the therapeutic effect of MSC-sIVs on the retinas of light-damaged mice, we collected the retinas of light-damaged mice (from Example 9) 7 days after MSC-sIVs treatment and performed RNA sequencing on the retinas of untreated (PBS-injected) light-damaged mice. The results showed significant differences in the following pathways: folate-mediated one-carbon unit metabolic pool, calcium signaling pathway, vascular smooth muscle contraction, cGMP-PKG signaling pathway, thiamine metabolism, cofactor biosynthesis, myocardial contraction and oxytocin signaling pathway, etc. (Figure 56).
4.2 RNA-seq结果显示MSC-sIVs对rd10小鼠视网膜变性的治疗作用4.2 RNA-seq results show the therapeutic effect of MSC-sIVs on retinal degeneration in rd10 mice
为了探究MSC-sIVs对rd10小鼠视网膜损伤的治疗作用，我们收集了经过MSC-sIVs治疗后7天的rd10小鼠视网膜（来自实施例10），与未经治疗（注射PBS）的rd10小鼠视网膜进行RNA-seq，结果显示以下通路存在显著差异：L1介导的囊泡运输再循环途径、L1CAM相互作用、IRE1alpha激活伴侣蛋白、XBP1(S)激活伴侣蛋白基因、未折叠蛋白反应(UPR)、帽结合复合物和eIFs结合后mRNA的激活、细胞-细胞外基质相互作用、向高尔基体的运输及其后续修饰、COPI介导的前向运输、内质网至高尔基体的前向运输和轴突导向等（图57）。In order to explore the therapeutic effect of MSC-sIVs on retinal damage in rd10 mice, we collected the retinas of rd10 mice (from Example 10) 7 days after MSC-sIVs treatment and performed RNA-seq on the retinas of untreated (PBS-injected rd10 mice). The results showed significant differences in the following pathways: L1-mediated vesicle transport recycling pathway, L1CAM interaction, IRE1alpha activation of chaperone proteins, XBP1(S) activation of chaperone protein genes, unfolded protein response (UPR), activation of mRNA after cap binding complex and eIFs binding, cell-extracellular matrix interaction, transport to the Golgi apparatus and its subsequent modification, COPI-mediated forward transport, forward transport from the endoplasmic reticulum to the Golgi apparatus, and axon guidance, etc. (Figure 57).
4.3蛋白质组学分析显示MSC-sIVs可以负调控内质网应激4.3 Proteomic analysis shows that MSC-sIVs can negatively regulate ER stress
本研究在蓝光损伤视网膜及rd10小鼠视网膜中证实MSC-sIVs在同等剂量下的治疗作用优于MSC-sEVs（参见实施例9和实施例10），为了进一步探究两种囊泡所含生物大分子在调控细胞代谢中的作用，我们对二者含有的总蛋白进行了基因集变异分析（Gene SetVariation Analysis, GSVA）通路富集分析。结果显示以下通路在MSC-sIVs中显著富集：内质网应激反应的负调控、内质网组分分泌颗粒、网格蛋白包被小泡、内质网与高尔基体之间的转运小泡、高尔基体到质膜的转运、CopI小泡和CopII小泡等。而细胞与细胞间的粘附、细胞与基质的粘附和底物粘附在MSC-sEVs中显著富集（图58）。This study confirmed that MSC-sIVs had a better therapeutic effect than MSC-sEVs at the same dose in blue light-damaged retina and rd10 mouse retina (see Examples 9 and 10). In order to further explore the role of biomacromolecules contained in the two vesicles in regulating cell metabolism, we performed Gene Set Variation Analysis (GSVA) pathway enrichment analysis on the total proteins contained in the two. The results showed that the following pathways were significantly enriched in MSC-sIVs: negative regulation of endoplasmic reticulum stress response, endoplasmic reticulum component secretory granules, clathrin-coated vesicles, transport vesicles between endoplasmic reticulum and Golgi apparatus, transport from Golgi apparatus to plasma membrane, CopI vesicles and CopII vesicles, etc. Cell-to-cell adhesion, cell-to-matrix adhesion and substrate adhesion were significantly enriched in MSC-sEVs (Figure 58).
4.4 miRNA-seq分析显示MSC-sIVs可以调控内质网稳态4.4 miRNA-seq analysis showed that MSC-sIVs can regulate endoplasmic reticulum homeostasis
为进一步分析MSC-sIVs较MSC-sEVs在改善视网膜损伤中的作用，在此，我们提取了在MSCs细胞的sEVs和sIVs中表达丰度前50的miRNA（图59），并对其进行了差异分析，选取了24个差异miRNA进行靶基因富集分析。结果如图60所示，这24个miRNA可以调控以下通路：内质网钙离子稳态、COPI囊泡包膜、蛋白酪氨酸激酶活性、蛋白激酶B信号、胶原蛋白分解代谢过程、STAT蛋白酪氨酸磷酸化的正调控、通过CpG岛甲基化对基因表达的负调控、内肽酶活性的负调控、有丝分裂纺锤体微管与着丝粒侧连接的正调控、DNA甲基转移酶活性、顶端皮层中枢神经系统形态发生等。To further analyze the role of MSC-sIVs in improving retinal damage compared with MSC-sEVs, we extracted the top 50 miRNAs expressed in sEVs and sIVs of MSCs cells (Figure 59), performed differential analysis, and selected 24 differential miRNAs for target gene enrichment analysis. The results are shown in Figure 60. These 24 miRNAs can regulate the following pathways: endoplasmic reticulum calcium ion homeostasis, COPI vesicle envelope, protein tyrosine kinase activity, protein kinase B signaling, collagen decomposition and metabolism, positive regulation of STAT protein tyrosine phosphorylation, negative regulation of gene expression through CpG island methylation, negative regulation of endopeptidase activity, positive regulation of mitotic spindle microtubules and centromere side connection, DNA methyltransferase activity, apical cortex central nervous system morphogenesis, etc.
4.5 Western Blot分析显示MSC-sIVs抑制光损伤小鼠视网膜内质网应激和凋亡4.5 Western Blot analysis showed that MSC-sIVs inhibited retinal endoplasmic reticulum stress and apoptosis in light-damaged mice
在本实施例4.3和4.4中我们揭示了MSC-sIVs可以作用于光损伤小鼠视网膜的钙离子信号通道，同时作用于rd10小鼠视网膜的内质网应激信号相关通路（UPR、XBP1和IRE1α）。另一方面，在针对MSC-sIVs与MSC-sEVs的蛋白质和miRNA的深入分析中，发现MSC-sIVs对内质网应激有负调控作用，且可以维持内质网稳态。因此我们使用Western blot进一步检测了光损伤条件下视网膜内质网应激相关蛋白的表达情况。葡萄糖调节蛋白78（Glucose-regulated protein 78, GRP78）与三个内质网应激传感器结合，GRP78表达水平上调是内质网应激最常用的标志物之一。因此我们取在蓝光照射前，照射后3天、5天和7天的小鼠视网膜，通过Western Blot检测GRP 78，结果显示与未照射小鼠相比，照射后第三天至第七天GRP78表达水平升高，其中第五天较为显著（图61）。以上结果提示：过度的蓝光暴露导致小鼠视网膜发生内质网应激，内质网应激可能是保护视网膜免受光损伤的治疗靶点。In Examples 4.3 and 4.4, we revealed that MSC-sIVs can act on the calcium ion signaling channels of the light-damaged mouse retina, and also act on the endoplasmic reticulum stress signaling-related pathways (UPR, XBP1, and IRE1α) of the retina of rd10 mice. On the other hand, in the in-depth analysis of proteins and miRNAs of MSC-sIVs and MSC-sEVs, it was found that MSC-sIVs had a negative regulatory effect on endoplasmic reticulum stress and could maintain endoplasmic reticulum homeostasis. Therefore, we used Western blot to further detect the expression of endoplasmic reticulum stress-related proteins in the retina under light damage conditions. Glucose-regulated protein 78 (GRP78) binds to three endoplasmic reticulum stress sensors, and the upregulation of GRP78 expression levels is one of the most commonly used markers of endoplasmic reticulum stress. Therefore, we took the mouse retinas before, 3 days, 5 days and 7 days after blue light irradiation, and detected GRP 78 by Western Blot. The results showed that compared with the unirradiated mice, the expression level of GRP78 increased from the third day to the seventh day after irradiation, and the fifth day was more significant (Figure 61). The above results suggest that excessive blue light exposure causes endoplasmic reticulum stress in the mouse retina, and endoplasmic reticulum stress may be a therapeutic target for protecting the retina from light damage.
在确认蓝光损伤小鼠视网膜存在内质网应激后，我们通过Western Blot检测相关靶蛋白，探究了不同剂量MSC-sEVs和MSC-sIVs对内质网应激通路的作用。结果显示与NoBL组相比，使用PBS治疗的光损伤组（BL-PBS）视网膜内质网应激相关蛋白的表达升高，表现为GRP78、p-PERK/PERK、IRE1a、ATF6、p-eIF2a和CHOP的水平升高，差异有统计学意义（p<0.05；图62）；与BL-PBS组相比低剂量和高剂量的MSC-sEVs治疗组内质网应激相关蛋白的表达水平没有显著差异；然而，与BL-PBS组相比低剂量的MSC-sIVs治疗组部分内质网应激相关蛋白的表达水平降低，表现为p-PERK/PERK和IRE1a的表达水平降低，差异有统计学意义（p<0.05）；高剂量的MSC-sIVs治疗组内质网应激相关蛋白的表达水平降低，表现为GRP78、p-PERK、ATF6、IRE1a、p-eIF2a/eIF2a和CHOP的表达水平降低，差异有统计学意义（p<0.05，图62）。与此同时，同剂量的sIVs较sEVs可以更显著的抑制GRP78和IRE1a的表达水平（p<0.05，图62）。以上结果提示：过度的蓝光照射导致视网膜发生内质网应激，MSC-sIVs通过抑制内质网应激保护视网膜免受光损伤。After confirming the presence of ER stress in the retina of blue light-damaged mice, we used Western Blot to detect related target proteins and explored the effects of different doses of MSC-sEVs and MSC-sIVs on the ER stress pathway. The results showed that compared with the NoBL group, the expression of retinal ER stress-related proteins in the light-damaged group (BL-PBS) treated with PBS was increased, as shown by increased levels of GRP78, p-PERK/PERK, IRE1a, ATF6, p-eIF2a and CHOP, and the difference was statistically significant (p<0.05; Figure 62); compared with the BL-PBS group, there was no significant difference in the expression levels of ER stress-related proteins in the low-dose and high-dose MSC-sEVs treatment groups; however, compared with the BL-PBS group The expression levels of some ER stress-related proteins in the low-dose MSC-sIVs treatment group were reduced, as shown by the reduced expression levels of p-PERK/PERK and IRE1a, with statistically significant differences (p<0.05); the expression levels of ER stress-related proteins in the high-dose MSC-sIVs treatment group were reduced, as shown by the reduced expression levels of GRP78, p-PERK, ATF6, IRE1a, p-eIF2a/eIF2a and CHOP, with statistically significant differences (p<0.05, Figure 62). At the same time, the same dose of sIVs can more significantly inhibit the expression levels of GRP78 and IRE1a than sEVs (p<0.05, Figure 62). The above results suggest that excessive blue light exposure causes ER stress in the retina, and MSC-sIVs protect the retina from light damage by inhibiting ER stress.
内质网应激最终结果导致细胞凋亡，接下来，我们检测了凋亡相关蛋白在视网膜中的表达水平。结果显示，与NoBL组相比，BL-PBS凋亡相关蛋白增加，表现为Cleaved-Caspase3和Bax表达水平升高，抗凋亡蛋白Bcl-2表达水平降低，差异有统计学意义（p<0.05；图63）；与BL-PBS组相比低剂量和高剂量的MSC-sEVs治疗组凋亡相关蛋白的表达水平没有显著差异；然而，与BL-PBS组相比高剂量的MSC-sIVs治疗组凋亡相关蛋白的表达水平降低，表现为Cleaved-Caspase3表达水平降低，促凋亡蛋白Bax的表达水平降低，抗凋亡蛋白Bcl-2表达水平升高，差异有统计学意义（p<0.05，图63）。同等剂量的MSC-sIVs较MSC-sEVs可以大幅提升抗凋亡蛋白Bcl-2的表达水平。以上结果提示：过度的蓝光照射导致视网膜发生凋亡，MSC-sIVs可以保护视网膜免受光损伤。Endoplasmic reticulum stress ultimately leads to cell apoptosis. Next, we detected the expression levels of apoptosis-related proteins in the retina. The results showed that compared with the NoBL group, the apoptosis-related proteins in the BL-PBS group increased, as shown by the increased expression levels of Cleaved-Caspase3 and Bax, and the decreased expression level of the anti-apoptotic protein Bcl-2, with statistically significant differences (p<0.05; Figure 63); compared with the BL-PBS group, there was no significant difference in the expression levels of apoptosis-related proteins in the low-dose and high-dose MSC-sEVs treatment groups; however, compared with the BL-PBS group, the expression levels of apoptosis-related proteins in the high-dose MSC-sIVs treatment group decreased, as shown by the decreased expression level of Cleaved-Caspase3, the decreased expression level of the pro-apoptotic protein Bax, and the increased expression level of the anti-apoptotic protein Bcl-2, with statistically significant differences (p<0.05, Figure 63). The same dose of MSC-sIVs can significantly increase the expression level of the anti-apoptotic protein Bcl-2 compared with MSC-sEVs. The above results suggest that excessive blue light exposure causes retinal apoptosis and MSC-sIVs can protect the retina from light damage.
4.6 Western Blot分析显示MSC-sIVs抑制rd10小鼠（p21）视网膜内质网应激和凋亡4.6 Western Blot analysis showed that MSC-sIVs inhibited retinal endoplasmic reticulum stress and apoptosis in rd10 mice (p21)
我们使用Western blot进一步检测了rd10小鼠不同发育阶段视网膜内质网应激相关蛋白的表达情况。我们取未干预条件下，rd10小鼠14天、21天和28天的视网膜，通过Western Blot检测GRP 78的表达水平，结果显示与相应周龄的WT小鼠相比，P14的视网膜GRP78的表达水平开始升高，p21和p28升高较为显著（图64）。以上结果提示：rd10小鼠视网膜发生内质网应激，内质网应激可能是保护rd10小鼠视网膜的潜在治疗靶点。We further detected the expression of retinal endoplasmic reticulum stress-related proteins in rd10 mice at different developmental stages using Western blot. We took the retinas of rd10 mice at 14, 21, and 28 days without intervention, and detected the expression level of GRP 78 by Western Blot. The results showed that compared with WT mice of the corresponding age, the expression level of GRP78 in the retina of P14 began to increase, and the increase was more significant at p21 and p28 (Figure 64). The above results suggest that retinal endoplasmic reticulum stress occurs in rd10 mice, and retinal endoplasmic reticulum stress may be a potential therapeutic target for protecting the retina of rd10 mice.
在确认rd10小鼠存在内质网应激后，我们通过Western Blot检测相关靶蛋白，探究了不同剂量MSC-sEVs和MSC-sIVs对内质网应激通路的作用。结果显示与WT组相比，p21的rd10小鼠视网膜内质网应激相关蛋白的表达升高，表现为GRP78、IRE1a、ATF6和CHOP的水平升高，差异有统计学意义（p<0.05；图65）；与rd10-PBS组相比MSC-sEVs治疗组视网膜内质网应激相关蛋白只有ATF6表达水平降低，其余标志蛋白的表达水平没有显著差异；然而，MSC-sIVs治疗组内质网应激相关蛋白的表达水平降低，表现为GRP78、IRE1a、ATF6和CHOP表达水平降低，差异有统计学意义（p<0.05；图65）。以上结果表明MSC-sIVs通过抑制内质网应激减少rd10小鼠视网膜损伤。After confirming that rd10 mice have ER stress, we used Western Blot to detect related target proteins and explored the effects of different doses of MSC-sEVs and MSC-sIVs on the ER stress pathway. The results showed that compared with the WT group, the expression of ER stress-related proteins in the retina of rd10 mice at p21 was increased, as shown by increased levels of GRP78, IRE1a, ATF6 and CHOP, with statistically significant differences (p<0.05; Figure 65); compared with the rd10-PBS group, only the expression level of ATF6 in the retinal ER stress-related proteins in the MSC-sEVs treatment group was reduced, and the expression levels of other marker proteins were not significantly different; however, the expression levels of ER stress-related proteins in the MSC-sIVs treatment group were reduced, as shown by decreased expression levels of GRP78, IRE1a, ATF6 and CHOP, with statistically significant differences (p<0.05; Figure 65). The above results indicate that MSC-sIVs reduce retinal damage in rd10 mice by inhibiting ER stress.
我们检测了凋亡相关蛋白在rd10小鼠视网膜中的表达水平。结果显示，与WT组相比，rd10小鼠在p21时视网膜凋亡相关蛋白增加，表现为Cleaved-Caspase3和Bax表达水平升高，抗凋亡蛋白Bcl-2表达水平降低，差异有统计学意义（p<0.05；图66）；与rd10-PBS组相比MSC-sEVs治疗组Cleaved-Caspase3表达水平降低，但是促凋亡蛋白Bax的表达水平仍然很高，抗凋亡蛋白Bcl-2表达水平并未显著升高。另一方面，MSC-sIVs治疗组视网膜凋亡相关蛋白的表达水平降低，表现为Cleaved-Caspase3表达水平降低，促凋亡蛋白Bax的表达水平降低，抗凋亡蛋白Bcl-2表达水平升高，差异有统计学意义（p<0.05，图66）。同等剂量的MSC-sIVs较MSC-sEVs可以大幅降低Caspase3的活化水平，降低促凋亡蛋白Bax的表达水平，同时提升抗凋亡蛋白Bcl-2的表达水平。以上结果表明，rd10小鼠在p21时视网膜发生凋亡，MSC-sIVs可以减少视网膜凋亡，其作用显著优于MSC-sEVs。We detected the expression levels of apoptosis-related proteins in the retina of rd10 mice. The results showed that compared with the WT group, the expression of apoptosis-related proteins in the retina of rd10 mice at p21 increased, as shown by increased expression levels of Cleaved-Caspase3 and Bax, and decreased expression levels of anti-apoptotic protein Bcl-2, with statistically significant differences (p<0.05; Figure 66); compared with the rd10-PBS group, the expression level of Cleaved-Caspase3 in the MSC-sEVs treatment group decreased, but the expression level of pro-apoptotic protein Bax was still high, and the expression level of anti-apoptotic protein Bcl-2 did not increase significantly. On the other hand, the expression levels of apoptosis-related proteins in the retina of the MSC-sIVs treatment group decreased, as shown by decreased expression levels of Cleaved-Caspase3, decreased expression levels of pro-apoptotic protein Bax, and increased expression levels of anti-apoptotic protein Bcl-2, with statistically significant differences (p<0.05, Figure 66). The same dose of MSC-sIVs can significantly reduce the activation level of Caspase3 and the expression level of the pro-apoptotic protein Bax, while increasing the expression level of the anti-apoptotic protein Bcl-2 compared with MSC-sEVs. The above results show that retinal apoptosis occurs in rd10 mice at p21, and MSC-sIVs can reduce retinal apoptosis, and its effect is significantly better than MSC-sEVs.
4.7 Western Blot分析显示MSC-sIVs抑制rd10小鼠（p28）视网膜内质网应激和凋亡4.7 Western Blot analysis showed that MSC-sIVs inhibited retinal endoplasmic reticulum stress and apoptosis in rd10 mice (p28)
通过图64确认rd10小鼠在p28时间点存在内质网应激，我们在p28时取小鼠视网膜，通过Western Blot继续检测内质网应激相关靶蛋白，探究MSC-sEVs和MSC-sIVs对内质网应激通路的作用。结果显示与WT组相比，rd10小鼠在p28时内质网应激相关蛋白的表达全面升高，表现为GRP78、p-PERK/PERK、IRE1a、ATF6、p-eIF2a/eIF2a和CHOP的表达水平升高，差异有统计学意义（p<0.05；图67）；与rd10-PBS组相比MSC-sEVs治疗组视网膜内质网应激相关蛋白的表达水平没有显著差异。然而，MSC-sIVs治疗组内质网应激相关蛋白的表达水平降低，表现为GRP78、p-PERK/PERK、IRE1a、ATF6、p-eIF2a/eIF2a和CHOP的表达水平降低，差异有统计学意义（p<0.05；图67）。以上结果表明MSC-sIVs通过抑制内质网应激减少rd10小鼠视网膜损伤，效果显著优于MSC-sEVs。Figure 64 confirmed that rd10 mice had ER stress at p28. We took the mouse retina at p28 and continued to detect ER stress-related target proteins by Western Blot to explore the effects of MSC-sEVs and MSC-sIVs on the ER stress pathway. The results showed that compared with the WT group, the expression of ER stress-related proteins in rd10 mice at p28 was generally increased, as shown by increased expression levels of GRP78, p-PERK/PERK, IRE1a, ATF6, p-eIF2a/eIF2a and CHOP, and the difference was statistically significant (p<0.05; Figure 67); compared with the rd10-PBS group, there was no significant difference in the expression level of ER stress-related proteins in the retina of the MSC-sEVs treatment group. However, the expression levels of ER stress-related proteins in the MSC-sIVs treatment group were reduced, as shown by the decreased expression levels of GRP78, p-PERK/PERK, IRE1a, ATF6, p-eIF2a/eIF2a and CHOP, with statistically significant differences (p<0.05; Figure 67). The above results indicate that MSC-sIVs can reduce retinal damage in rd10 mice by inhibiting ER stress, and the effect is significantly better than MSC-sEVs.
我们检测了凋亡相关蛋白在rd10小鼠视网膜中的表达水平。结果显示，与WT组相比，rd10小鼠在p28时视网膜凋亡相关蛋白增加，表现为Cleaved-Caspase3和Bax表达水平升高，抗凋亡蛋白Bcl-2表达水平降低，差异有统计学意义（p<0.05；图68）；与rd10-PBS组相比MSC-sEVs治疗组对凋亡相关蛋白无显著治疗作用。MSC-sIVs治疗组视网膜凋亡相关蛋白的表达水平降低，表现为Cleaved-Caspase3表达水平降低，促凋亡蛋白Bax的表达水平降低，抗凋亡蛋白Bcl-2表达水平升高，差异有统计学意义（p<0.05，图68）。同等剂量的MSC-sIVs较MSC-sEVs可以大幅降低Caspase3的活化水平，降低促凋亡蛋白Bax的表达水平，同时提升抗凋亡蛋白Bcl-2的表达水平。以上结果表明，rd10小鼠在p28时视网膜发生凋亡，MSC-sIVs可以抑制rd10小鼠视网膜凋亡信号，促进抗凋亡蛋白表达，其作用显著优于MSC-sEVs。We detected the expression levels of apoptosis-related proteins in the retina of rd10 mice. The results showed that compared with the WT group, the retinal apoptosis-related proteins of rd10 mice increased at p28, as shown by the increased expression levels of Cleaved-Caspase3 and Bax, and the decreased expression level of the anti-apoptotic protein Bcl-2, with statistically significant differences (p<0.05; Figure 68); compared with the rd10-PBS group, the MSC-sEVs treatment group had no significant therapeutic effect on apoptosis-related proteins. The expression levels of retinal apoptosis-related proteins in the MSC-sIVs treatment group were reduced, as shown by the decreased expression level of Cleaved-Caspase3, the decreased expression level of the pro-apoptotic protein Bax, and the increased expression level of the anti-apoptotic protein Bcl-2, with statistically significant differences (p<0.05, Figure 68). The same dose of MSC-sIVs can significantly reduce the activation level of Caspase3, the expression level of the pro-apoptotic protein Bax, and the expression level of the anti-apoptotic protein Bcl-2 compared with MSC-sEVs. The above results indicate that retinal apoptosis occurs in rd10 mice at p28, and MSC-sIVs can inhibit retinal apoptosis signals in rd10 mice and promote the expression of anti-apoptotic proteins, which is significantly better than MSC-sEVs.
5、小结5. Summary
在本实施例中，我们深入探讨了MSC-sIVs治疗视网膜损伤的分子机制。通过对视网膜的RNA-seq分析，我们在光损伤小鼠视网膜中发现了钙离子信号通路的富集，而在rd10小鼠视网膜中观察到了内质网应激信号通路的显著富集。为了进一步揭示MSC-sIVs的蛋白表达特征，我们采用了GSVA方法，结果显示MSC-sIVs能够负调控内质网应激。通过对MSC-sIVs中高丰度的miRNA进行富集分析，我们发现这些miRNA在调控内质网稳态方面发挥关键作用。在后续实验中，我们观察到光损伤小鼠视网膜和rd10小鼠视网膜中内质网应激信号的显著高表达。为了验证MSC-sIVs的治疗效果，我们利用Western Blot方法检测了治疗后的视网膜内质网应激信号的表达水平。结果表明， MSC-sIVs对内质网应激信号的抑制效果显著，并能够有效减少视网膜凋亡。In this example, we explored the molecular mechanism of MSC-sIVs in treating retinal damage. Through RNA-seq analysis of the retina, we found enrichment of calcium ion signaling pathways in the retina of light-damaged mice, and observed significant enrichment of endoplasmic reticulum stress signaling pathways in the retina of rd10 mice. To further reveal the protein expression characteristics of MSC-sIVs, we used the GSVA method, and the results showed that MSC-sIVs could negatively regulate endoplasmic reticulum stress. By enrichment analysis of highly abundant miRNAs in MSC-sIVs, we found that these miRNAs play a key role in regulating endoplasmic reticulum homeostasis. In subsequent experiments, we observed significantly high expression of endoplasmic reticulum stress signals in the retina of light-damaged mice and rd10 mice. In order to verify the therapeutic effect of MSC-sIVs, we used the Western Blot method to detect the expression level of retinal endoplasmic reticulum stress signals after treatment. The results showed that MSC-sIVs had a significant inhibitory effect on endoplasmic reticulum stress signals and could effectively reduce retinal apoptosis.
当内质网面临压力，例如蛋白质的合成、折叠或转运受到干扰时，会激发一系列信号通路来帮助恢复细胞稳定状态或者引导细胞凋亡。在应激状态下，钙离子会从内质网释放到细胞质中，从而触发钙离子依赖的信号传递。与此同时，内质网膜上的IRE1α蛋白会被激活，它能剪切XBP1的mRNA，生成具有转录活性的XBP1(S)。这种活性形式进一步促进伴侣蛋白基因的表达，这些伴侣蛋白对蛋白质的正确处理至关重要。此外，作为一种适应性反应，未折叠蛋白反应（UPR）通过激活内质网跨膜蛋白如IRE1α、PERK和ATF6等，来调节转录和翻译过程，以缓解内质网应激。这些机制和通路协同工作，以维护内质网的稳态，保护细胞免受应激损伤，促进细胞的适应性和生存能力。视网膜可以将光信号转化为电信号，是耗氧量最高的组织之一，糖尿病，过度的光照，缺血缺氧等都会导致视网膜发生氧化应激，伴随内质网应激的发生。When the ER is under stress, such as when protein synthesis, folding or transport is disturbed, a series of signaling pathways are stimulated to help restore cell homeostasis or guide cell apoptosis. Under stress, calcium ions are released from the ER into the cytoplasm, triggering calcium-dependent signaling. At the same time, the IRE1α protein on the ER membrane is activated, which can cleave XBP1 mRNA to generate transcriptionally active XBP1(S). This active form further promotes the expression of chaperone protein genes, which are essential for the correct processing of proteins. In addition, as an adaptive response, the unfolded protein response (UPR) regulates transcription and translation processes to relieve ER stress by activating ER transmembrane proteins such as IRE1α, PERK and ATF6. These mechanisms and pathways work together to maintain ER homeostasis, protect cells from stress damage, and promote cell adaptability and survival. The retina can convert light signals into electrical signals and is one of the tissues with the highest oxygen consumption. Diabetes, excessive light, ischemia and hypoxia can all cause oxidative stress in the retina, accompanied by the occurrence of ER stress.
有研究显示，光暴露可以在体外环境中引发光感受器细胞和视网膜色素上皮细胞（RPE细胞）的应激反应，这包括氧化应激、内质网应激和自噬。在光损伤的小鼠视网膜中，也观察到了内质网应激的现象。而且，抑制内质网应激有助于减少光诱导的光感受器细胞凋亡，这表明内质网应激可能是治疗视网膜损伤疾病的一个重要靶点。MSC-sIVs可以抑制内质网应激信号，这一发现为MSC-sIVs治疗视网膜氧化应激及损伤等疾病提供了有力支持。Studies have shown that light exposure can induce stress responses in photoreceptor cells and retinal pigment epithelial cells (RPE cells) in vitro, including oxidative stress, endoplasmic reticulum stress, and autophagy. Endoplasmic reticulum stress has also been observed in the light-damaged mouse retina. Moreover, inhibiting endoplasmic reticulum stress helps reduce light-induced photoreceptor cell apoptosis, suggesting that endoplasmic reticulum stress may be an important target for the treatment of retinal damage diseases. MSC-sIVs can inhibit endoplasmic reticulum stress signals, a finding that provides strong support for MSC-sIVs to treat diseases such as retinal oxidative stress and damage.
实施例12：MSC-sIVs促进人角膜上皮细胞增殖、趋化、迁移Example 12: MSC-sIVs promote proliferation, chemotaxis and migration of human corneal epithelial cells
1、实验方法1. Experimental methods
1.1 实验试剂1.1 Experimental reagents
表18实验试剂Table 18 Experimental reagents
1.2人角膜上皮细胞的培养1.2 Culture of human corneal epithelial cells
人角膜上皮细胞（human corneal epithelial cells，HCEC）是由武汉普诺赛生命科技有限公司提供的，是一种上皮细胞样的贴壁细胞。使用的完全培养基为：DMEM/F12(Invitrogen，USA)+15%FBS(Invitrogen)+5μg/ml Insulin(procell，PBI80332)+10μg/mlhuman EGF(gibco, PHG0311)+1%P/S。在37°C的5%CO2培养箱中培养。冻存条件：55%基础培养基+40%FBS+5%DMSO。Human corneal epithelial cells (HCEC) were provided by Wuhan Pronocell Life Science Co., Ltd. and are epithelial-like adherent cells. The complete culture medium used was: DMEM/F12 (Invitrogen, USA) + 15% FBS (Invitrogen) + 5μg/ml Insulin (procell, PBI80332) + 10μg/ml human EGF (gibco, PHG0311) + 1% P/S. Cultured in a 5% CO2 incubator at 37°C. Cryopreservation conditions: 55% basal culture medium + 40% FBS + 5% DMSO.
细胞复苏：将冷冻管中的细胞迅速解冻至于37℃水浴，与完全培养基1:10稀释，离心1000g 5min，细胞重悬后，转移进含有完全培养基5mL的T25培养瓶中培养细胞。Cell recovery: Thaw the cells in the cryovial rapidly in a 37°C water bath, dilute with complete medium 1:10, centrifuge at 1000g for 5 min, resuspend the cells, and transfer them into a T25 culture flask containing 5 mL of complete medium for culture.
传代步骤：每次吸出原培养液后加入PBS润洗细胞并丢弃，加入1mL 0.25%胰蛋白酶溶液（含EDTA）消化细胞，显微镜下见细胞完成消化后加入3mL完全培养基终止消化。转移收集细胞悬液离心 1000g 5min，离心完成后吸出上清丢弃，完全培养基重悬转移至新培养瓶。每隔2-3天更换一次培养基。当细胞融合达到80%时，以1:2-1:3的继代比例进行传代，并使用P3至P5的细胞进行后续实验。Subculture steps: After aspirating the original culture medium each time, add PBS to rinse the cells and discard them. Add 1mL of 0.25% trypsin solution (containing EDTA) to digest the cells. After the cells are digested under the microscope, add 3mL of complete culture medium to stop digestion. Transfer and collect the cell suspension and centrifuge at 1000g for 5min. After centrifugation, aspirate and discard the supernatant, resuspend in complete culture medium and transfer to a new culture bottle. Change the culture medium every 2-3 days. When the cell fusion reaches 80%, subculture at a subculture ratio of 1:2-1:3, and use cells from P3 to P5 for subsequent experiments.
1.3CCK-8检测细胞增殖1.3CCK-8 detection of cell proliferation
将HCEC接种到96孔板中，稀释1倍的完全培养基中饥饿处理培养，生长至60%汇合。加入不同浓度梯度的sIVs或sEVs（实施例2制备）（2.5、5、10、20、40μg/ml）。在24小时、48小时后用CCK-8试剂盒赋予30min在酶标仪在450nm处测量样品吸光度。以24小时、48小时的对照组在450nm的吸光度为基线，计算sIVs或sEVs（20、40μg/ml）的相对吸光度，绘制该浓度sIVs或sEVs对HCEC细胞的增殖作用曲线。HCEC were inoculated into 96-well plates, starved and cultured in a 1-fold dilution of complete medium, and grown to 60% confluence. Different concentration gradients of sIVs or sEVs (prepared in Example 2) (2.5, 5, 10, 20, 40 μg/ml) were added. After 24 hours and 48 hours, the CCK-8 kit was used to give 30 minutes and the sample absorbance was measured at 450nm on a microplate reader. The relative absorbance of sIVs or sEVs (20, 40 μg/ml) was calculated using the absorbance of the control group at 450nm for 24 hours and 48 hours as the baseline, and the proliferation effect curve of sIVs or sEVs at this concentration on HCEC cells was plotted.
1.4 Transwell检测sIVs和sEVs作用后HCEC趋化作用1.4 Transwell assay for HCEC chemotaxis after sIVs and sEVs
将Transwell室用含有1%P/S的DF-12基础培养基激活，30min后用1%P/S的DF-12基础培养基HCEC。实验组下室中加入不同浓度梯度的sIVs或sEVs（2.5、5、10、20、40μg/ml）。孵育24小时后，附着在过滤膜上表面的细胞被去除。迁移的细胞用4%戊二醛固定，并用0.5%结晶紫染色。细胞在倒置显微镜下计数。用ImageJ对照片上的细胞进行处理和计数。The Transwell chamber was activated with DF-12 basal medium containing 1% P/S, and 30 min later, HCEC was added with DF-12 basal medium containing 1% P/S. Different concentration gradients of sIVs or sEVs (2.5, 5, 10, 20, 40 μg/ml) were added to the lower chamber of the experimental group. After incubation for 24 h, the cells attached to the upper surface of the filter membrane were removed. The migrated cells were fixed with 4% glutaraldehyde and stained with 0.5% crystal violet. The cells were counted under an inverted microscope. The cells in the photos were processed and counted using ImageJ.
1.5 划痕实验检测sIVs和sEVs作用后HCEC迁移作用1.5 Scratch assay to detect HCEC migration after sIVs and sEVs
使用6孔板预先标记，后将HCECs铺板并生长至80-90%铺满时，用尺子固定后，使用无菌的1mL移液枪尖尖端在孔板底部划出一个笔直均匀的划痕，对治疗组加入不同浓度梯度的sIVs或sEVs（2.5、5、10、20、40μg/ml），对照组加入同等体积的PBS。在划痕后0h，6h，12h，24h使用荧光显微镜记录伤口闭合情况，并使用ImageJ软件进行测量。伤口闭合比率=[无细胞区域（0小时）-无细胞区域（目标小时）]/无细胞区域（0小时）×100％。A 6-well plate was pre-marked, and HCECs were plated and grown to 80-90% confluence. After fixing with a ruler, a straight and uniform scratch was made on the bottom of the well plate using a sterile 1 mL pipette tip. Different concentration gradients of sIVs or sEVs (2.5, 5, 10, 20, 40 μg/ml) were added to the treatment group, and the same volume of PBS was added to the control group. Wound closure was recorded using a fluorescence microscope at 0h, 6h, 12h, and 24h after scratching, and measured using ImageJ software. Wound closure ratio = [cell-free area (0 hour) - cell-free area (target hour)] / cell-free area (0 hour) × 100%.
2、实验结果2. Experimental results
2.1 MSC-sIVs在体外促进HCEC的增殖2.1 MSC-sIVs promote the proliferation of HCEC in vitro
CCK-8实验结果显示，在24小时后不同浓度梯度的sIVs均能显著促进HCEC的增殖，sEVs与对照组无明显差异（图69）。sIVs促进HCEC增殖的效果在作用24小时后均显著强于sEVs（图70）。在48小时后不同浓度梯度的sIVs均能显著促进HCEC的增殖，且具有浓度依赖性，仅40μg/ml sEVs能促进HCEC的增殖，sEVs余浓度与对照组无明显差异（图71）。sIVs促进HCEC增殖的效果在作用48小时后均显著强于sEVs（图72）。随后我们选取相同的有效作用浓度（20μg/ml和40μg/ml）的sIVs和sEVs作用于HCEC并对HECE的增殖情况进行动态检测。20μg/ml sIVs在24、48小时作用后均能显著促进HECE的增殖，比同浓度sEVs作用显著（图73）。40μg/ml sIVs在24、48小时作用后均能显著促进HECE的增殖，比同浓度sEVs作用显著（图74）。The results of the CCK-8 experiment showed that after 24 hours, sIVs of different concentration gradients could significantly promote the proliferation of HCEC, and there was no significant difference between sEVs and the control group (Figure 69). The effect of sIVs in promoting HCEC proliferation was significantly stronger than sEVs after 24 hours (Figure 70). After 48 hours, sIVs of different concentration gradients could significantly promote the proliferation of HCEC, and it was concentration-dependent. Only 40μg/ml sEVs could promote the proliferation of HCEC, and the remaining concentrations of sEVs had no significant difference from the control group (Figure 71). The effect of sIVs in promoting HCEC proliferation was significantly stronger than sEVs after 48 hours (Figure 72). Then we selected sIVs and sEVs of the same effective concentration (20μg/ml and 40μg/ml) to act on HCEC and dynamically detect the proliferation of HECE. 20μg/ml sIVs could significantly promote the proliferation of HECE after 24 and 48 hours, which was more significant than sEVs of the same concentration (Figure 73). 40μg/ml sIVs could significantly promote the proliferation of HECE after 24 and 48 hours of action, which was more significant than the same concentration of sEVs (Figure 74).
2.2 MSC-sIVs在体外促进HCEC的趋化作用2.2 MSC-sIVs promote chemotaxis of HCEC in vitro
Transwell实验结果显示，在24小时后浓度为5、10、20、40μg/ml的sIVs和sEVs能浓度依赖性促进HCEC的趋化作用，通过小室的细胞数量显著高于对照组（图75），同浓度下sIVs的作用效果比sEVs显著（图76）。The results of the Transwell experiment showed that after 24 hours, sIVs and sEVs at concentrations of 5, 10, 20, and 40 μg/ml could promote the chemotaxis of HCEC in a concentration-dependent manner, and the number of cells passing through the chamber was significantly higher than that of the control group (Figure 75). At the same concentration, the effect of sIVs was more significant than that of sEVs (Figure 76).
2.3 MSC-sIVs在体外促进HCEC的划痕修复2.3 MSC-sIVs promote HCEC scratch repair in vitro
划痕实验结果显示sIVs及sEVs能显著促进HCEC的迁移（图77），在12、18小时sIVs的促进迁移的能力比sEVs更显著（图78）。The results of the scratch experiment showed that sIVs and sEVs could significantly promote the migration of HCEC (Figure 77). At 12 and 18 hours, the ability of sIVs to promote migration was more significant than that of sEVs (Figure 78).
3. 小结3. Summary
与MSC-sEVs相比，MSC-sIVs可以在体外促进HCEC增殖、趋化、划痕修复，有效改善细胞损伤，预期其可有效改善因角膜损伤导致的角膜疾病，具有修复角膜上皮损伤的潜力。Compared with MSC-sEVs, MSC-sIVs can promote HCEC proliferation, chemotaxis, and scratch repair in vitro, effectively improving cell damage. It is expected that it can effectively improve corneal diseases caused by corneal damage and has the potential to repair corneal epithelial damage.
实施例13：MSC-sIVs促进小鼠角膜创伤模型角膜上皮基质的修复Example 13: MSC-sIVs promote the repair of corneal epithelial stroma in a mouse corneal trauma model
1、实验方法1. Experimental methods
1.1角膜创伤模型的构建1.1 Construction of corneal trauma model
角膜创伤模型：使用了C57BL/6和小鼠（6-8周龄）在实验动物中心以无菌条件下饲养。手术前一天予氧氟沙星滴眼液点眼3次/日。小鼠经腹腔注射麻醉后，使用复方托比卡胺滴眼液散瞳，表面麻醉后，棉签吸去多余水分，充分暴露小鼠角膜，在角膜中央使用2毫米环钻标记（未到小鼠角膜缘），用AlgerBrush II角膜去除器划伤整个角膜上皮和前基质。术后使用抗生素凝胶点眼。Corneal trauma model: C57BL/6 and mice (6-8 weeks old) were used and raised under sterile conditions in the experimental animal center. Ofloxacin eye drops were applied 3 times a day the day before surgery. After intraperitoneal injection of anesthesia, the mice were dilated with compound tropicamide eye drops. After surface anesthesia, the excess water was absorbed with a cotton swab, the mouse cornea was fully exposed, a 2 mm trephine mark was used in the center of the cornea (not reaching the mouse corneal limbus), and the entire corneal epithelium and anterior stroma were scratched with an AlgerBrush II corneal remover. Antibiotic gel was applied to the eyes after surgery.
1.2 角膜创伤模型角膜上皮和基质对sEVs和sIVs的亲和力1.2 Affinity of corneal epithelium and stroma for sEVs and sIVs in corneal trauma model
MSCs来源的sIVs与sEVs（实施例2制备）使用膜标记染料DiD标记染色。将角膜创伤造模小鼠使用标记后的sIVs与sEVs点眼，4小时后用4%多聚甲醛固定。使用OCT进行包埋，保存于-80℃冰箱，进行冰冻切片后在激光共聚焦显微镜下检测内吞作用。MSCs-derived sIVs and sEVs (prepared in Example 2) were labeled and stained with the membrane-labeling dye DiD. The corneal trauma model mice were eye-dotted with labeled sIVs and sEVs, and fixed with 4% paraformaldehyde 4 hours later. They were embedded in OCT and stored in a -80°C refrigerator. After frozen sections, endocytosis was detected under a laser confocal microscope.
1.3 同浓度（0.1μg/ml）MSC-sIVs及MSC-sEVs对小鼠角膜创伤治疗1.3 Treatment of corneal trauma in mice with the same concentration (0.1 μg/ml) of MSC-sIVs and MSC-sEVs
小鼠角膜创伤模型造模后，分为PBS组、0.1μg/μl sIVs治疗组及0.1μg/μl sEVs治疗组进行治疗。一天两次，每次3.5μl。After the corneal trauma model was established, the mice were divided into PBS group, 0.1μg/μl sIVs treatment group and 0.1μg/μl sEVs treatment group for treatment, twice a day, 3.5μl each time.
1.3荧光素钠染色示角膜上皮缺损范围1.3 Fluorescein sodium staining shows the extent of corneal epithelial defect
将裂隙灯生物显微镜调整至视野清晰，将小鼠调整到最佳位置观察眼表。小鼠角膜上皮荧光素钠染色：0.1%荧光素钠溶液4μL，染色10-15s后用棉签擦去多余荧光素钠溶液。使用ImageJ统计角膜上皮缺损面积及角膜面积。角膜损伤闭合比率=角膜上皮缺损面积（t小时）/角膜面积（t小时）×100%。Adjust the slit lamp biomicroscope to a clear field of view, and adjust the mouse to the best position to observe the ocular surface. Mouse corneal epithelial fluorescein sodium staining: 0.1% sodium fluorescein solution 4μL, after staining for 10-15s, wipe off the excess sodium fluorescein solution with a cotton swab. Use ImageJ to count the corneal epithelial defect area and corneal area. Corneal injury closure ratio = corneal epithelial defect area (t hour)/corneal area (t hour) × 100%.
2、实验结果2. Experimental results
2.1 角膜损伤部位对MSC-sIVs的吸收率高于MSC-sEVs2.1 The absorption rate of MSC-sIVs in the corneal injury site is higher than that of MSC-sEVs
使用DiD标记后等量的MSC-sIVs和MSC-sEVs点眼，4小时后取眼球进行冰冻切片，结果如图79所示，红色为DiD标记的囊泡，蓝色为细胞核，点眼后的囊泡大多分布于角膜损伤的断端，角膜上皮断端对MSC-sIVs的内吞优于MSC-sEVs；MSC-sIVs分布于暴露的角膜基质，而MSC-sEVs基本没有停留在角膜基质。这说明角膜的损伤部位对MSC-sIVs的吸收率更高，MSC-sIVs可以较长时间停留在角膜损伤部位，发挥治疗作用，具有极高的应用潜力。Equal amounts of MSC-sIVs and MSC-sEVs were labeled with DiD and then applied to the eyes. Four hours later, the eyeballs were taken for frozen sections. The results are shown in Figure 79. The red color is the DiD-labeled vesicles, and the blue color is the cell nucleus. The vesicles after eye application are mostly distributed at the broken ends of corneal damage. The broken ends of corneal epithelium are better at internalizing MSC-sIVs than MSC-sEVs. MSC-sIVs are distributed in the exposed corneal stroma, while MSC-sEVs basically do not stay in the corneal stroma. This indicates that the damaged part of the cornea has a higher absorption rate for MSC-sIVs, and MSC-sIVs can stay at the damaged part of the cornea for a longer time to play a therapeutic role, which has extremely high application potential.
2.2 MSC-sIVs显著促进小鼠角膜上皮缺损愈合2.2 MSC-sIVs significantly promote the healing of corneal epithelial defects in mice
使用等量的MSC-sIVs和MSC-sEVs进行点眼治疗，在治疗前及治疗后第1天，第2天进行荧光素钠染色显示角膜缺损部位。代表性裂隙灯图片及荧光素钠染色图片如图80所示，实验结果（图81）显示，MSC-sIVs和MSC-sEVs与对照组相比能显著促进角膜伤口伤口愈合的作用，在治疗后第1天显示MSC-sIVs比MSC-sEVs的治疗效果更显著。Equal amounts of MSC-sIVs and MSC-sEVs were used for eye drop treatment, and sodium fluorescein staining was performed before treatment and on the first and second days after treatment to show the corneal defect site. Representative slit lamp images and sodium fluorescein staining images are shown in Figure 80. The experimental results (Figure 81) show that MSC-sIVs and MSC-sEVs can significantly promote the healing of corneal wounds compared with the control group, and the therapeutic effect of MSC-sIVs is more significant than that of MSC-sEVs on the first day after treatment.
实施例14：MSC-sIVs促进角膜损伤后角膜敏感性恢复Example 14: MSC-sIVs promotes corneal sensitivity recovery after corneal injury
1、实验方法1. Experimental methods
角膜创伤模型的构建同实施例13，角膜知觉计示角膜敏感性，使用Cochet-Bonnet感觉仪（Luneau Ophtalmologie，Chartres Cedex，France）来评估角膜敏感性。纤维单丝尖端用于接触角膜，单丝的长度从 6.0厘米到 0.5厘米不等，以 0.5厘米为单位，直到找到角膜敏感性阈值。角膜敏感性阈值被定义为超过50%的尝试次数（5次尝试中至少3次或更多次）导致角膜眨眼反射的刺激。一旦单丝的长度达到阈值（最大长度6厘米），将测试尝试次数从5次增加到10次，以提高其准确性。The construction of the corneal trauma model is the same as in Example 13. The corneal sensory meter shows corneal sensitivity. The Cochet-Bonnet sensory meter (Luneau Ophtalmologie, Chartres Cedex, France) is used to evaluate corneal sensitivity. The tip of the fiber monofilament is used to contact the cornea. The length of the monofilament varies from 6.0 cm to 0.5 cm, in units of 0.5 cm, until the corneal sensitivity threshold is found. The corneal sensitivity threshold is defined as more than 50% of the attempts (at least 3 or more times out of 5 attempts) leading to the stimulation of the corneal blink reflex. Once the length of the monofilament reaches the threshold (maximum length 6 cm), the number of test attempts is increased from 5 to 10 to improve its accuracy.
2、实验结果2. Experimental results
在治疗后第7天和第14天进行角膜知觉计检测角膜敏感性阈值，结果显示（图82），在治疗后第7天MSC-sIVs能显著促进角膜敏感性恢复，在治疗第14天MSC-sIVs及MSC-sEVs均显著促进角膜敏感性恢复，MSC-sIVs作用更显著。Corneal sensitivity threshold was detected by corneal perceptometer on the 7th and 14th days after treatment. The results showed (Figure 82) that MSC-sIVs could significantly promote the recovery of corneal sensitivity on the 7th day after treatment, and both MSC-sIVs and MSC-sEVs could significantly promote the recovery of corneal sensitivity on the 14th day after treatment, with the effect of MSC-sIVs being more significant.
3、小结3. Summary
MSC-sIVs可以快速修复角膜上皮损伤，促进角膜敏感性恢复，其疗效优于MSC-sEVs，具有极大的临床应用潜力。MSC-sIVs can quickly repair corneal epithelial damage and promote the recovery of corneal sensitivity. Its therapeutic effect is better than MSC-sEVs and it has great potential for clinical application.
实施例15：MSC-sIVs促进角膜碱烧伤后角膜敏感性恢复Example 15: MSC-sIVs promotes corneal sensitivity recovery after corneal alkali burn
1、实验方法1. Experimental methods
1.1角膜碱烧伤模型的构建1.1 Construction of corneal alkali burn model
角膜碱烧伤模型：使用了C57BL/6和小鼠（6-8周龄）在实验动物中心以无菌条件下饲养。手术前一天予氧氟沙星滴眼液点眼3次/日。小鼠经腹腔注射麻醉后，使用复方托比卡胺滴眼液散瞳，表面麻醉后，棉签吸去多余水分，充分暴露小鼠角膜，使用直径2毫米的滤纸浸入1M的氢氧化钠溶液，随后吸去多余水分，将滤纸放置在在角膜中央1分钟后使用生理盐水冲洗眼表1分钟。术后使用抗生素凝胶点眼。Corneal alkali burn model: C57BL/6 and mice (6-8 weeks old) were used and raised under sterile conditions in the experimental animal center. Ofloxacin eye drops were applied 3 times a day the day before surgery. After intraperitoneal injection of anesthesia, the mice were dilated with compound tropicamide eye drops. After surface anesthesia, cotton swabs were used to absorb excess water, fully expose the mouse cornea, and filter paper with a diameter of 2 mm was immersed in 1M sodium hydroxide solution, and then excess water was absorbed. The filter paper was placed in the center of the cornea for 1 minute and then the ocular surface was rinsed with saline for 1 minute. Antibiotic gel was used for eye drops after surgery.
1.2使用裂隙灯拍摄角膜新生血管1.2 Using a slit lamp to photograph corneal neovascularization
在造模前，造模后3天，7天，10天和14天，分别拍摄小鼠角膜侧面的裂隙灯照片，显示角膜缘后侧的血管，及长入角膜的新生血管。Slit lamp photographs of the side of the mouse cornea were taken before modeling, 3 days, 7 days, 10 days and 14 days after modeling, showing the blood vessels behind the corneal limbus and the new blood vessels growing into the cornea.
1.3角膜铺片显示角膜新生血管1.3 Corneal flat mount showing corneal neovascularization
在造模后14天脱颈处死小鼠，取眼球后使用PFA固定2小时，取角膜后在PFA中4℃固定过夜，随后加入封闭液，室温2小时，一抗CD31(R&D)，4℃过夜，随后清洗一抗6小时；二抗驴抗羊IgG-594(abcam)，4℃过夜，随后清洗二抗6小时；随后使用抗荧光衰减封片剂封片。使用共聚焦显微镜拍摄。14 days after modeling, mice were killed by dislocation of the neck, and the eyeballs were fixed with PFA for 2 hours. The corneas were fixed in PFA at 4°C overnight, followed by addition of blocking solution at room temperature for 2 hours, primary antibody CD31 (R&D), 4°C overnight, followed by washing the primary antibody for 6 hours; secondary antibody donkey anti-sheep IgG-594 (abcam), 4°C overnight, followed by washing the secondary antibody for 6 hours; then anti-fluorescence attenuation mounting medium was used to seal the slides. Confocal microscopy was used for photography.
2、实验结果2. Experimental results
图83显示碱烧伤组小鼠角膜自第三天起，血管增粗，自第七天至第十四天，角膜新生血管发出自角巩膜缘的血管，进入角膜，对照组在第十四天时，可见角膜自角膜缘至中央被新生血管侵袭。图84所示可以直观的看到新生血管侵袭角膜组织的范围，经统计，sIVs治疗可以显著降低新生血管的侵袭，其效果优于sEVs（图85）。Figure 83 shows that the cornea of mice in the alkali burn group had thickened blood vessels since the third day, and from the seventh to the fourteenth day, new blood vessels in the cornea were emitted from the limbus of the cornea and entered the cornea. On the fourteenth day in the control group, the cornea was invaded by new blood vessels from the limbus to the center. Figure 84 shows the scope of the new blood vessels invading the corneal tissue. According to statistics, sIVs treatment can significantly reduce the invasion of new blood vessels, and its effect is better than sEVs (Figure 85).
3、小结3. Summary
MSC-sIVs可以减少角膜新生血管，其疗效优于MSC-sEVs，具有极大的临床应用潜力。MSC-sIVs can reduce corneal neovascularization, and its therapeutic effect is better than MSC-sEVs, which has great potential for clinical application.
在本发明中，通过实施例1-3，我们分离富集了sIVs并且优化了分离参数，得到了最佳分离方案，实施例4-7在物理性质和所含生物大分子方面对比了sIVs与现有技术广泛报道的sEVs的区别，表明sIVs是细胞内独特的囊泡群，显著区别于sEVs，其中实施例5还证实MSCs的sIVs具有独特的蛋白质表达谱。实施例8通过体内体外实验证实，细胞和视网膜组织对MSC-sIVs具有很好的亲和力，且优于MSC-sEVs。实施例9-10展示了我们在两种小鼠视网膜损伤模型（蓝光照射引起的视网膜损伤模型和rd10视网膜色素变性遗传模型）中评估了MSC-sIVs和MSC-sEVs的治疗效果，发现MSC-sIVs在保护视网膜光感受器细胞方面表现出更优异的疗效，具有更好的治疗效果。实施例11证实MSC-sIVs通过抑制内质网应激信号发挥对视网膜的保护作用。实施例12-14证实MSC-sIVs可以在体外促进角膜上皮细胞生长，在角膜损伤模型中，可以促进角膜上皮和基质修复。实施例15证实MSC-sIVs可以抑制病理性角膜新生血管生长。综上，成体干细胞产生的sIVs可以治疗视网膜变性损伤及角膜损伤等眼科疾病，其效果优于sEVs。本发明的成体干细胞来源的细胞内纳米囊泡在医药领域中具有非常好的应用和研究价值。In the present invention, through Examples 1-3, we separated and enriched sIVs and optimized the separation parameters to obtain the best separation scheme. Examples 4-7 compared the differences between sIVs and sEVs widely reported in the prior art in terms of physical properties and contained biomacromolecules, indicating that sIVs are a unique vesicle population in cells, which is significantly different from sEVs. Example 5 also confirmed that MSCs sIVs have a unique protein expression profile. Example 8 confirmed through in vivo and in vitro experiments that cells and retinal tissues have a good affinity for MSC-sIVs, and are superior to MSC-sEVs. Examples 9-10 show that we evaluated the therapeutic effects of MSC-sIVs and MSC-sEVs in two mouse retinal damage models (retinal damage model caused by blue light irradiation and rd10 retinitis pigmentosa genetic model), and found that MSC-sIVs showed better efficacy in protecting retinal photoreceptor cells and had better therapeutic effects. Example 11 confirmed that MSC-sIVs protects the retina by inhibiting endoplasmic reticulum stress signals. Examples 12-14 confirmed that MSC-sIVs can promote corneal epithelial cell growth in vitro, and can promote corneal epithelial and matrix repair in a corneal injury model. Example 15 confirmed that MSC-sIVs can inhibit pathological corneal neovascularization. In summary, sIVs produced by adult stem cells can treat ophthalmic diseases such as retinal degeneration and corneal injury, and its effect is better than sEVs. The intracellular nanovesicles derived from adult stem cells of the present invention have very good application and research value in the field of medicine.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.
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