技术领域technical field
本发明属于角膜离体组织的保存技术领域，具体涉及一种角膜组织中长期立体保存营养胶囊及其制备方法。The invention belongs to the technical field of corneal isolated tissue preservation, and in particular relates to a medium- and long-term three-dimensional corneal tissue preservation nutritional capsule and a preparation method thereof.
背景技术Background technique
角膜病是我国第二位致盲眼病，视力障碍患者大部分是因角膜失明而致残，其次是角膜功能障碍导致。由于角膜损伤具有不可逆性，因此角膜移植是目前恢复视力的主要手段。但医用角膜材料因来源有限，角膜组织活性保存受保存液种类和保存时间的限制，而需要角膜移植的患者需求量较大，因此角膜供体材料一直存在严重匮乏的情形。同时角膜移植也存在着短板，因角膜供体数量和质量受到限制，阻碍了外科手术的发展，此外，可能出现感染、移植失败和免疫反应等并发症。Corneal disease is the second leading cause of blindness in my country. Most visually impaired patients are disabled due to corneal blindness, followed by corneal dysfunction. Because corneal damage is irreversible, corneal transplantation is currently the main means of restoring vision. However, due to the limited sources of medical corneal materials, the active preservation of corneal tissue is limited by the type and storage time of the preservation solution, and the demand for corneal transplantation is large, so there has been a serious shortage of corneal donor materials. At the same time, there are also shortcomings in corneal transplantation, because the quantity and quality of corneal donors are limited, which hinders the development of surgical operations. In addition, complications such as infection, graft failure, and immune response may occur.
人们已经对角膜组织的结构进行了深入研究，研究表明角膜组织结构层次分明，自外向内可分为五层：上皮层、前弹力层、基质层、后弹力层和内皮层。目前用于角膜移植的角膜保存技术主要集中在如何维持内皮的活性和完整性，延长内皮细胞的活性保存时间，而对于角膜基质的保存研究较少。People have conducted in-depth research on the structure of corneal tissue. Studies have shown that the structure of corneal tissue is well-defined and can be divided into five layers from outside to inside: epithelial layer, Bowman's membrane, stroma layer, Bowman's membrane and endothelial layer. The current corneal preservation technology for corneal transplantation mainly focuses on how to maintain the activity and integrity of the endothelium and prolong the preservation time of the endothelial cells, but there are few studies on the preservation of the corneal stroma.
《国际眼科杂志》中综述文献《角膜保存方法现状及进展》提及了现有角膜保存的常用方法，其将角膜保存分为短期保存、中期保存、长期保存和超长期保存。(1)对于短期保存而言，湿房保存2d后内皮细胞的活性将下降50％；(2)对于中期保存而言，使用角膜保存液可维持内皮细胞活性达到4～14d，并随着保存技术的改进以及角膜保存液的成分不断改变，角膜内皮细胞的活性也逐渐提高；(3)对于长期保存而言，器官培养法虽然能够较好保存内皮细胞的活性，并使得移植后排斥率大大降低，但其对设备及检测要求较高、操作复杂、易发生感染，且费用较高，在我国现阶段环境下较难推广；(4)对于超长期保存而言，甘油保存法属于无活性的角膜保存法，保存的角膜一般仅用于做板层移植或角膜溃疡穿孔和破裂伤时短时间无法获得新鲜角膜材料的急诊角膜修补术，较少用于穿透性角膜移植术；而深低温保存法一般是将角膜在冷冻剂保护下，降低温度到-80℃，再贮存于-196℃液氮中，使角膜内皮细胞处于“休眠状态”，使用时进行适当的复温处理，恢复角膜活性，但是该方法保存的设备昂贵，过程复杂，技术要求较高，而且复温时易造成角膜内皮细胞损伤。可见，现有角膜保存方法无论时间长短均着重于维持内皮细胞的活性和完整性，对于角膜基质的保存效果，现有的保存方法较少涉及。The review document "Current Status and Progress of Corneal Preservation Methods" in "International Journal of Ophthalmology" mentioned the common methods of existing corneal preservation, which divided corneal preservation into short-term preservation, medium-term preservation, long-term preservation and ultra-long-term preservation. (1) For short-term storage, the activity of endothelial cells will decrease by 50% after 2 days of wet room storage; (2) For medium-term storage, the use of corneal preservation solution can maintain the activity of endothelial cells for 4-14 days, and with the preservation With the improvement of technology and the constant change of the composition of corneal preservation solution, the activity of corneal endothelial cells has gradually increased; (3) For long-term preservation, although the organ culture method can better preserve the activity of endothelial cells, it will greatly increase the rejection rate after transplantation. However, it has high requirements for equipment and detection, complex operation, easy infection, and high cost, so it is difficult to promote in China's current environment; (4) For ultra-long-term preservation, glycerin preservation method is inactive The preserved cornea is generally only used for lamellar transplantation or emergency keratoplasty when fresh corneal materials cannot be obtained in a short period of time when corneal ulcer perforation and rupture are used, and it is less used for penetrating keratoplasty; The cryopreservation method is generally to lower the temperature of the cornea to -80°C under the protection of cryogen, and then store it in liquid nitrogen at -196°C, so that the corneal endothelial cells are in a "dormant state". Corneal activity, but the preservation equipment of this method is expensive, the process is complicated, the technical requirements are high, and the corneal endothelial cells are easily damaged during rewarming. It can be seen that the existing corneal preservation methods focus on maintaining the activity and integrity of endothelial cells regardless of the length of time, and the existing preservation methods are less involved in the preservation effect of corneal stroma.
角膜基质由角膜基质细胞(HCSCs)组成，HCSCs是起源于神经嵴的间充质细胞，这些细胞夹在板层之间，维持板层结缔组织的基质成分，高度组织化的胶原板层提供了角膜透明所需的机械支持和生物物理特性。因此角膜基质的纤维结构和透明度的维持，以及角膜基质细胞活性的长时间维持，是决定其保存质量的关键因素。The corneal stroma is composed of corneal stromal cells (HCSCs), mesenchymal cells originating from the neural crest, which are sandwiched between the lamellae and maintain the stromal component of the lamellar connective tissue, and the highly organized collagenous lamina provides Mechanical support and biophysical properties required for corneal transparency. Therefore, the maintenance of the fibrous structure and transparency of the corneal stroma, as well as the long-term maintenance of the activity of corneal stromal cells, are the key factors that determine the quality of its preservation.
飞秒激光小切口角膜基质透镜取出术(Small Incision Lenticule Extraction，SMILE)是近年来角膜屈光手术领域快速发展的典型代表，其作为一种安全、有效、微创的近视矫治激光手术技术，获得了世界范围内的认可。SMILE手术中在角膜基质层内取出的透镜组织称为“角膜基质透镜”，SMILE角膜基质透镜来源非常丰富，全球80多个国家已开展SMILE手术超600万例/眼，并可产生数以万计的人角膜基质透镜，我国每年开展SMILE手术也达到一百万台以上。然而SMILE手术中取出的这部分“角膜基质透镜”往往作为手术废弃物被丢弃，并未得到有效利用。Femtosecond laser small incision lenticule extraction (SMILE) is a typical representative of the rapid development of corneal refractive surgery in recent years. As a safe, effective and minimally invasive laser surgical technique for myopia correction, it has won recognized worldwide. The lens tissue taken out of the corneal stroma layer in SMILE surgery is called "corneal stroma lens". The source of SMILE corneal stroma lens is very rich. Over 6 million cases of SMILE surgery have been carried out in more than 80 countries around the world, and tens of thousands of them can be produced. The total number of human corneal stromal lenses, and the number of SMILE operations carried out in my country every year has reached more than one million. However, the part of the "corneal stromal lens" removed during the SMILE operation is often discarded as surgical waste and has not been effectively utilized.
角膜基质透镜是角膜组织的一部分，由有序排列的胶原构成，因为其无血管和淋巴组织，移植时发生免疫排斥反应的概率极小，因此可再利用作为一种良好的角膜供体材料，可用于矫正远视、近视、老视等屈光不正和用于治疗圆锥角膜、角膜营养不良、角膜溃疡、角膜穿孔、边缘角膜变性(周边角膜的免疫性、非感染性疾病)和角膜基质变薄等角膜疾病，以及利用其构建组织工程化生物角膜的载体等。Corneal stromal lens is a part of corneal tissue, which is composed of collagen arranged in an orderly manner. Because it has no blood vessels and lymphatic tissue, the probability of immune rejection during transplantation is extremely small, so it can be reused as a good corneal donor material. It can be used to correct hyperopia, myopia, presbyopia and other refractive errors and to treat keratoconus, corneal dystrophy, corneal ulcer, corneal perforation, marginal corneal degeneration (immune and non-infectious diseases of peripheral cornea) and corneal stroma thinning Corneal diseases such as cornea, as well as the carrier for constructing tissue engineered biocornea using it.
近年来，一些学者开展了将原本废弃的“角膜基质透镜”变废为宝的研究，临床初步结果表明SMILE透镜植入后具有优异的生物相容性和安全性，具有非常好的应用前景。然而，这些研究采用的SMILE透镜仅涉及从捐献者提供的新鲜角膜组织中获得，或是经过深低温冷冻保存后的角膜中获得，而并未对角膜基质透镜进行中长期保存后再进行移植。对于这类大量手术废弃物的再利用而言，能否有效维持角膜基质透镜的纤维结构和透明度，以及如何长时间维持角膜基质细胞的活性，是角膜基质透镜中长期保存过程中首要解决的问题。In recent years, some scholars have carried out research on turning the discarded "corneal stromal lens" into treasure. The preliminary clinical results show that the SMILE lens has excellent biocompatibility and safety after implantation, and has a very good application prospect. However, the SMILE lenses used in these studies were only obtained from fresh corneal tissue provided by donors, or from corneas after cryopreservation, and the corneal stromal lenses were not stored for medium and long-term before transplantation. For the reuse of such a large amount of surgical waste, whether the fiber structure and transparency of the corneal stromal lens can be effectively maintained, and how to maintain the activity of the corneal stromal cells for a long time is the primary problem to be solved during the long-term preservation of the corneal stromal lens. .
然而，现有关于角膜基质透镜保存方法的研究，仅涉及对角膜透镜进行超低温长期保存，而对于在4℃下采用保存液进行中期保存的活性并不佳且保存时间较短。However, the existing research on corneal stromal lens preservation methods only involves ultra-low temperature long-term preservation of corneal lenses, and the medium-term preservation of the preservation solution at 4°C is not very active and the preservation time is short.
中国专利文献CN 111066777 A公开了一种角膜透镜超低温长期保存方法，其是将角膜透镜保存液无菌分装至无菌冻存管后，将角膜透镜转移至冻存管中，然后放置到-60℃～-80℃深低温冰箱进行程序降温并过夜，再放置到-196℃液氮中进行长期保存。该方法可用于SMILE手术收集的角膜透镜的长期保存，但其方法复杂，采用的设备昂贵，无法进行推广应用，同时并不能对角膜基质细胞的活性进行长期维持，而是使角膜基质细胞处于“休眠状态”来进行长期保存。Chinese patent document CN 111066777 A discloses a method for ultra-low temperature long-term storage of corneal lenses, which is to aseptically pack the corneal lens preservation solution into sterile cryopreservation tubes, then transfer the corneal lenses into the cryopreservation tubes, and then place them in- 60 ℃ ~ -80 ℃ deep-low temperature refrigerator for program cooling and overnight, and then placed in -196 ℃ liquid nitrogen for long-term storage. This method can be used for long-term preservation of corneal lenses collected by SMILE surgery, but the method is complicated, the equipment used is expensive, and it cannot be popularized and applied. At the same time, it cannot maintain the activity of corneal stromal cells for a long time, but keeps the corneal stromal cells in " Hibernate state" for long-term storage.
叶青等在其文献《Smile来源的角膜基质透镜构建组织工程角膜基质支架的实验研究》中记载，从Smile来源的角膜基质透镜中分离培养人角膜基质细胞，采用MTT法检测人纤维蛋白粘合剂(FS)对细胞的毒性作用，将FS粘贴双层角膜基质透镜构建的双层透镜支架分别置于不同介质(无水甘油、透明质酸钠、胎牛血清、模拟湿房环境)及不同温度(常温、4℃、-20℃)中保存，其研究结果发明：4℃保存14d后，角膜基质透镜支架出现一定程度的开裂现象，且影响其透明度，而湿房环境下角膜基质透镜支架除出现开裂外，还出现了严重的皱缩和水肿现象，最终得出将角膜基质透镜支架在-20℃无水甘油中保存的效果较好，能够维持透镜支架的稳定性和透明度。这一研究结果表明，常规的角膜保存液在4℃的保存条件下难以实现角膜基质透镜的中期保存。Ye Qing et al. recorded in their literature "Experimental Research on the Construction of Tissue-Engineered Corneal Structural Scaffolds by Smile-derived Corneal Stromal Lens" that human corneal stromal cells were isolated and cultured from Smile-derived corneal stromal lenses, and human fibrin adhesion was detected by MTT assay. To investigate the toxicity of FS to cells, the double-layer lens holder constructed by FS pasted double-layer corneal stromal lens was placed in different media (anhydrous glycerin, sodium hyaluronate, fetal bovine serum, simulated wet room environment) and different Preserved at room temperature (normal temperature, 4°C, -20°C), the research results found that after 14 days of storage at 4°C, the corneal stroma lens holder cracked to a certain extent, which affected its transparency, while the corneal stroma lens holder in the wet room environment In addition to cracking, severe shrinkage and edema also appeared. Finally, it was concluded that the preservation effect of the corneal stroma lens holder in -20°C anhydrous glycerin can maintain the stability and transparency of the lens holder. The results of this study indicate that it is difficult to achieve mid-term preservation of corneal stromal lenses with conventional corneal preservation solutions at 4°C.
上述报道表明，目前常用的角膜保存方法(如中期保存中的常用保存液：Optisol液)虽然对于内皮细胞的活性维持起到了较好效果，但对于角膜基质透镜的保存，特别是对角膜基质细胞活性的维持，仍存在较大不足。主要存在以下缺点：(1)常规的保存液仅适用于角膜的中期低温保存，保存时间最长只有14天，时间越长会导致细胞的形态及活性异常，不利于角膜透镜组织的长期运输；(2)保存后角膜厚度会变薄，缺乏对透镜胶原纤维结构和透明性的维持；(3)中期保存液以细胞培养基作为基础液，为角膜组织提供所需的营养物质，但不能满足角膜基质细胞生理代谢所需的全部营养物质；这些缺点均限制了角膜基质透镜的再利用及推广。The above reports show that although the commonly used corneal preservation methods (such as the commonly used preservation solution in medium-term preservation: Optisol solution) have a good effect on maintaining the activity of endothelial cells, they are not effective for the preservation of corneal stromal lenses, especially for corneal stromal cells. The maintenance of the activity still has big deficiencies. The main disadvantages are as follows: (1) The conventional preservation solution is only suitable for medium-term low-temperature preservation of the cornea, and the longest preservation time is only 14 days. The longer the time, the abnormal shape and activity of the cells will be caused, which is not conducive to the long-term transportation of corneal lens tissue; (2) The thickness of the cornea will become thinner after preservation, lacking the maintenance of the collagen fiber structure and transparency of the lens; (3) The medium-term preservation solution uses the cell culture medium as the base solution to provide the corneal tissue with the required nutrients, but it cannot meet the All the nutrients required for the physiological metabolism of corneal stromal cells; these shortcomings limit the reuse and promotion of corneal stromal lenses.
因此，如何实现中长期保持角膜基质透镜的胶原纤维结构和透明性，以及如何实现中长期保持角膜基质细胞的活性，提供一种角膜基质或透镜组织的保存方法，为角膜基质透镜的中长期保存以及长途运输提供可能，成为亟待解决的技术问题。Therefore, how to maintain the collagen fiber structure and transparency of the corneal stromal lens in the medium and long term, and how to maintain the activity of the corneal stromal cells in the medium and long term, provide a preservation method for the corneal stroma or lens tissue, and provide a medium and long-term preservation method for the corneal stromal lens. And the possibility of long-distance transportation has become a technical problem to be solved urgently.
发明内容Contents of the invention
本发明的目的就是为了解决上述技术问题，从而提供一种角膜组织中长期立体保存营养胶囊及其制备方法。本发明的技术目的在于：一方面解决现有的角膜保存液大多仅适用于内皮细胞的中期保存，存在对于角膜基质透镜的保存时间较短，无法实现角膜基质透镜的中长期保存和长途运输的问题；另一方面解决现有的角膜保存液存在难以保持角膜基质透镜的胶原纤维结构和透明度，以及无法长时间维持角膜基质细胞活性的问题。The object of the present invention is to solve the above-mentioned technical problems, thereby providing a nutritional capsule for medium- and long-term three-dimensional preservation of corneal tissue and a preparation method thereof. The technical purpose of the present invention is: on the one hand, it solves the problem that most of the existing corneal preservation solutions are only suitable for medium-term preservation of endothelial cells, and the preservation time for corneal stroma lenses is relatively short, so that medium and long-term preservation and long-distance transportation of corneal stroma lenses cannot be realized. Problem; on the other hand, it is difficult to maintain the collagen fiber structure and transparency of the corneal stromal lens in the existing corneal preservation solution, and it is impossible to maintain the activity of corneal stromal cells for a long time.
为了实现上述技术目的，本发明采用了如下的技术方案：In order to realize above-mentioned technical purpose, the present invention adopts following technical scheme:
本发明首先提供了一种角膜组织中长期立体保存营养胶囊，该营养胶囊为内部含营养液的胶囊球结构，所述胶囊球结构从外到内依次包括脂质模拟层、水液模拟层和润滑层，所述脂质模拟层包括海藻酸钠溶液与钙离子通过扩散配位络合形成的外壳层，所述水液模拟层包括角膜基础培养基和营养物质；所述润滑层包括硫酸软骨素。The present invention firstly provides a nutritional capsule for long-term stereoscopic preservation of corneal tissue. The nutritional capsule is a capsule ball structure containing a nutrient solution inside. The capsule ball structure includes a lipid simulation layer, a water liquid simulation layer and Lubricating layer, the lipid-mimicking layer includes a shell layer formed by sodium alginate solution and calcium ion through diffusion coordination complexation, the water-liquid-mimicking layer includes corneal basal medium and nutrients; the lubricating layer includes sulfated cartilage white.
本发明提供的上述营养胶囊，在用于保存角膜基质透镜时，对保存在胶囊壳内部的角膜基质透镜能够起到很好的物理阻隔和缓冲作用，能够实现中期和长期维持胶原纤维的结构和透明度，同时能够很好保持角膜基质细胞的活性，其活性保持效果优于Optisol保存液和甘油，活性保持时间长达至少28天。另外在长途运输过程中，本发明的营养胶囊用于保存角膜组织时，不会因为外力造成角膜组织的损伤，其保存效果显著优于现有的角膜保存液。本发明的营养胶囊不仅适用于角膜基质透镜的保存，同时能够适用于保存其它角膜组织。The above nutrition capsule provided by the present invention, when used to preserve the corneal stroma lens, can play a good physical barrier and buffer role for the corneal stroma lens stored inside the capsule shell, and can maintain the structure and structure of collagen fibers in the medium and long term. Transparency, and at the same time, it can well maintain the activity of corneal stromal cells. Its activity maintenance effect is better than Optisol preservation solution and glycerin, and the activity maintenance time is as long as at least 28 days. In addition, during long-distance transportation, when the nutritional capsule of the present invention is used to preserve corneal tissue, it will not cause damage to corneal tissue due to external force, and its preservation effect is significantly better than the existing corneal preservation solution. The nutritional capsule of the invention is not only suitable for the preservation of corneal stroma lenses, but also suitable for the preservation of other corneal tissues.
人体仿生学研究表明，人体器官自然体液微环境是组织最好的保存液，其能够持续不断地为组织细胞提供新鲜的培养液，保持细胞维持充足稳定的营养，同时不断地排除代谢废物使细胞生长在相对稳定的环境中，保持较高的细胞活性和组织完整结构。因此，一种理想的角膜组织保存液需具备以下特点：1)能够模拟天然角膜组织外体液，满足细胞生理代谢所需营养物质，防止细胞发生生理性损伤；2)能够维持角膜器官的组织形态和生理结构；3)能够为长途运输角膜组织提供立体缓冲，避免因外力带来移植器官或组织的结构性损伤。Human bionics studies have shown that the natural body fluid microenvironment of human organs is the best preservation fluid for tissues, which can continuously provide fresh culture fluid for tissue cells, maintain sufficient and stable nutrition for cells, and continuously eliminate metabolic waste to make cells Growing in a relatively stable environment, maintaining high cell activity and tissue integrity. Therefore, an ideal corneal tissue preservation solution needs to have the following characteristics: 1) It can simulate the extracorporeal fluid of natural corneal tissue, meet the nutrients required by the physiological metabolism of cells, and prevent physiological damage to cells; 2) It can maintain the tissue shape of corneal organs and physiological structure; 3) It can provide three-dimensional cushioning for long-distance transportation of corneal tissue, and avoid structural damage to transplanted organs or tissues caused by external forces.
本发明人在将海藻酸钠与钙离子进行快速扩散配位时，创造性地得到了一种立体缓冲胶囊球外壳，该胶囊球外壳具有很好的立体结构，能够自动成型。在此基础上，本发明人进一步对营养胶囊的结构进行了大量研究，经过反复摸索后，最终设计出了营养胶囊从外到内依次包括脂质模拟层、水液模拟层和润滑层的三层结构，实验结果表明，这三层结构与人体泪液结构组成高度相似，能够很好模拟人体泪液的功能，实现角膜组织的中长期高效保存。The present inventor creatively obtained a three-dimensional buffer capsule spherical shell when rapidly diffusing and coordinating sodium alginate and calcium ions. The capsule spherical shell has a good three-dimensional structure and can be automatically formed. On this basis, the inventor further conducted a large amount of research on the structure of the nutritional capsule, and after repeated explorations, finally designed a three-dimensional structure of the nutritional capsule that includes a lipid simulation layer, a water simulation layer and a lubricating layer from the outside to the inside. The experimental results show that the three-layer structure is highly similar to the structure of human tears, which can well simulate the function of human tears and realize the medium and long-term efficient preservation of corneal tissue.
本发明设计的营养胶囊结构为外部具备实体凝胶外壳，内部空心结构中填充营养液的球形结构，其中由海藻酸钠与钙离子通过扩散配位络合形成的立体凝胶层外壳具有空隙；通过加入硫酸软骨素钠、透明质酸钠和右旋糖酐等物质作为营养保存液添加到胶囊球内部，该水液模拟层可以通过胶囊球外壳的空隙进行营养供给、物质代谢并阻断病原体；最内层由硫酸软骨素组成，其可以与保存在营养液(即水液模拟层)内部的角膜基质透镜表面产生静电作用形成润滑层，发挥很好的润滑功能。本发明按照上述方法构建出了一种新型的能够与人体泪液结构组成高度相似的营养胶囊，可很好用于角膜组织的中长期立体保存，起到优良的物理阻隔、营养供给和表面润滑等多重作用。经过长达至少28天的立体保存后，角膜基质透镜的胶原纤维结构和透明度能够很好保持，同时角膜基质细胞的活性也能够高度维持。The nutrition capsule structure designed by the present invention has a solid gel shell on the outside and a spherical structure filled with nutrient solution in the hollow inside, wherein the three-dimensional gel layer shell formed by diffusion and coordination of sodium alginate and calcium ions has gaps; By adding chondroitin sulfate sodium, sodium hyaluronate, dextran and other substances into the inside of the capsule ball as a nutrient preservation solution, the water liquid simulation layer can supply nutrients, metabolize substances and block pathogens through the gaps in the shell of the capsule ball; the innermost The layer is composed of chondroitin sulfate, which can generate electrostatic interaction with the surface of the corneal stroma lens stored in the nutrient solution (ie, the water simulated layer) to form a lubricating layer, and play a good lubricating function. According to the above method, the present invention constructs a new type of nutritional capsule that can be highly similar to human tears in structure and composition, which can be well used for medium and long-term three-dimensional preservation of corneal tissue, and plays an excellent role in physical barrier, nutrient supply and surface lubrication, etc. Multiple roles. After at least 28 days of stereoscopic storage, the collagen fiber structure and transparency of the corneal stromal lens can be well maintained, and the activity of the corneal stromal cells can also be maintained at a high level.
而当本发明人将该营养胶囊制备成实心之后，如对比例1所示，发现营养胶囊对角膜基质透镜的保存效果明显变差，角膜基质细胞的活性下降明显，将不利于对角膜基质透镜的中长期保存。And after the present inventor prepared this nutrition capsule into solid, as shown in comparative example 1, find that the preservation effect of nutrition capsule to corneal stroma lens obviously becomes worse, the activity of corneal stroma cell declines obviously, will be unfavorable to corneal stroma lens medium and long-term storage.
本发明提供的营养胶囊，与现有的角膜保存液相比，保存周期可延长到至少28天，很好实现了对角膜组织的长期保存和长途运输的目的，同时立体胶囊壳结构能够对保存在内部的角膜组织起到很好的物理阻隔和缓冲作用，即使是在长途运输过程中，也不会因为外力造成角膜组织的损伤，其保存效果显著优于现有的角膜保存液。Compared with the existing corneal preservation solution, the nutritional capsule provided by the present invention can extend the preservation period to at least 28 days, and realizes the purpose of long-term preservation and long-distance transportation of corneal tissue, and the three-dimensional capsule shell structure can be used for preservation. The internal corneal tissue plays a very good physical barrier and buffer role. Even during long-distance transportation, the corneal tissue will not be damaged by external force, and its preservation effect is significantly better than the existing corneal preservation solution.
将本发明的营养胶囊用于长期保存角膜组织透镜(保存达28天)后，再将该保存后的角膜组织透镜用于角膜透镜移植手术，治疗角膜营养不良、圆锥角膜等疾病获得了很好的临床效果，其安全性和有效性均较佳。由此可以看出，本发明为角膜基质透镜的保存、运输和再利用提供了一种简单、安全、可控的优化策略。同时，本发明的营养胶囊还能够用于其它角膜离体组织或器官的中长期保存，其效果要优于现有的角膜保存液。After the nutrient capsule of the present invention is used for long-term storage of corneal tissue lenses (preservation reaches 28 days), the corneal tissue lenses after this preservation are used for corneal lens transplantation, and the treatment of diseases such as corneal dystrophy and keratoconus has achieved good results. The clinical effect, its safety and efficacy are better. It can be seen that the present invention provides a simple, safe and controllable optimization strategy for the preservation, transportation and reuse of corneal stromal lenses. At the same time, the nutrition capsule of the present invention can also be used for medium and long-term preservation of other corneal isolated tissues or organs, and its effect is better than that of the existing corneal preservation solution.
进一步的是，本发明的角膜组织立体保存营养胶囊的球直径大小为1-20mm，营养胶囊的外壳厚度为10-500μm。上述尺寸范围能够很好满足临床所需角膜组织的保存需求，同时本发明的营养胶囊的尺寸大小和外壳层厚度可以根据实际需要进行个性化调整，均是能够在工艺上实现和控制的。Furthermore, the spherical diameter of the nutrition capsule for three-dimensional preservation of corneal tissue of the present invention is 1-20 mm, and the shell thickness of the nutrition capsule is 10-500 μm. The above size range can well meet the clinical needs of corneal tissue preservation, and at the same time, the size and thickness of the outer shell layer of the nutritional capsule of the present invention can be adjusted individually according to actual needs, which can be realized and controlled in the process.
进一步的是，需要保存的角膜组织是保存于营养胶囊的胶囊球外壳层以内。所述角膜组织包括角膜基质、基质透镜、角膜内皮或角膜上皮。本发明所述角膜组织包括角膜基质透镜，还可以包括其它的角膜离体组织，如角膜基质、角膜内皮和角膜上皮等，同时也包括其它的离体器官组织。一般角膜组织保存在水液模拟层内或是润滑层。Further, the corneal tissue that needs to be preserved is preserved in the outer shell layer of the capsule ball of the nutrition capsule. The corneal tissue includes corneal stroma, stromal lens, corneal endothelium or corneal epithelium. The corneal tissue in the present invention includes corneal stroma lens, and may also include other isolated corneal tissues, such as corneal stroma, corneal endothelium, corneal epithelium, etc., and also includes other isolated organ tissues. Generally, the corneal tissue is preserved in the water simulated layer or the lubricating layer.
进一步的是，本发明的角膜组织立体保存营养胶囊中的营养物质包括硫酸软骨素钠、透明质酸钠和/或右旋糖酐等，还可以添加其它角膜保存液中常用的营养物质，如海藻酸钠、抗坏血酸钠、海藻糖、葡聚糖、聚乙烯醇、壳聚糖、聚乙二醇等，可以灵活选用营养物质。Further, the nutrients in the corneal tissue stereopreservation nutrition capsule of the present invention include sodium chondroitin sulfate, sodium hyaluronate and/or dextran, etc., and other nutrients commonly used in corneal preservation solutions, such as sodium alginate, can also be added , sodium ascorbate, trehalose, dextran, polyvinyl alcohol, chitosan, polyethylene glycol, etc., can flexibly choose nutrients.
进一步的是，本发明的角膜组织立体保存营养胶囊中添加的角膜基础培养基，可以包括DMEM/F-12、DMEM、MEM或DME，以及可以在培养基中添加其它成分，如双抗等。Furthermore, the corneal basal medium added in the nutrition capsule for three-dimensional preservation of corneal tissue of the present invention may include DMEM/F-12, DMEM, MEM or DME, and other components such as double antibodies may be added to the medium.
本发明还提供了上述角膜组织中长期立体保存营养胶囊的制备方法，其包括以下步骤：The present invention also provides a preparation method for the long-term three-dimensional preservation of nutritional capsules in the above-mentioned corneal tissue, which includes the following steps:
(1)向角膜基础培养基中添加硫酸软骨素，然后加入营养物质混合，形成水液模拟层和润滑层；(1) Add chondroitin sulfate to the corneal basal medium, then add nutrients and mix to form a water-liquid simulated layer and a lubricating layer;
(2)向步骤(1)所得物中加入海藻酸钠，形成预凝胶溶液；(2) adding sodium alginate to the resultant of step (1) to form a pregel solution;
(3)向预凝胶溶液中加入钙离子，成胶后，形成脂质模拟层，即得所述角膜组织中长期立体保存营养胶囊。(3) Calcium ions are added to the pre-gel solution to form a lipid mimic layer after forming a gel to obtain the nutritional capsule for long-term three-dimensional preservation of the corneal tissue.
更为具体的，本发明提供的一种营养胶囊的制备方法如下：More specifically, the preparation method of a kind of nutritional capsule provided by the invention is as follows:
(1)以角膜基础培养基作为母液，向母液中加入质量分数为0.1％～3％的硫酸软骨素、1％双抗、0.01％～0.5％抗坏血酸钠和0.2％～2％海藻糖，上述物质溶解完全后得到水液模拟层和润滑层；(1) Taking the corneal basal culture medium as the mother solution, adding chondroitin sulfate, 1% double antibody, 0.01% to 0.5% sodium ascorbate and 0.2% to 2% trehalose with a mass fraction of 0.1% to 3% in the mother solution, the above After the substance is completely dissolved, the water-liquid simulation layer and the lubricating layer are obtained;
(2)向步骤(1)所得物中加入海藻酸钠至其终浓度为0.5wt％～3wt％，形成预凝胶溶液；(2) adding sodium alginate to the resultant of step (1) to a final concentration of 0.5wt% to 3wt%, forming a pregel solution;
(3)向预凝胶溶液中滴加入50～300mM CaCl2水溶液，经10～280s成胶后，即得所述角膜组织中长期立体保存营养胶囊。(3) Adding 50-300 mM CaCl2 aqueous solution dropwise to the pre-gel solution, and gelling for 10-280 s, to obtain the nutritional capsule for long-term three-dimensional preservation of the corneal tissue.
本发明将含有海藻酸钠的营养保存液与钙离子快速扩散配位形成立体缓冲胶囊，而硫酸软骨素与角膜基质透镜表面产生静电作用形成润滑层，可起到立体保存和营养供给作用。经实验发现，采用成胶时间在10～280s之间，可以很好获得脂质模拟层、水液模拟层和润滑层的三层结构，达到与人体泪液结构组成高度相似。In the present invention, the nutrient preservation solution containing sodium alginate is quickly diffused and coordinated with calcium ions to form a three-dimensional buffer capsule, and the chondroitin sulfate and the surface of the corneal stroma lens generate electrostatic interaction to form a lubricating layer, which can play the role of three-dimensional preservation and nutrition supply. It has been found through experiments that the three-layer structure of the lipid simulation layer, the water simulation layer and the lubricating layer can be well obtained by using the gelation time between 10 and 280s, which is highly similar to the structure and composition of human tears.
进一步的是，本发明的步骤(2)中所述预凝胶溶液中海藻酸钠的浓度为1wt％。经本发明人研究发现，步骤(2)中采用该浓度下的海藻酸钠溶液所得预凝胶的黏度最佳，获得的营养胶囊的支撑性最好，同时获得的立体球形胶囊壳的均匀性最好。采用其它浓度的海藻酸钠也能够获得相应的营养胶囊，但其支撑性和胶囊壳的均匀性均不如在1％浓度下更佳。Further, the concentration of sodium alginate in the pregel solution in step (2) of the present invention is 1 wt%. The inventors have found that the viscosity of the pregel obtained by using the sodium alginate solution at this concentration in step (2) is the best, and the support of the obtained nutritional capsules is the best, and the uniformity of the three-dimensional spherical capsule shell obtained at the same time most. The corresponding nutritional capsules can also be obtained by using other concentrations of sodium alginate, but the support and the uniformity of the capsule shell are not as good as those at 1% concentration.
进一步的是，步骤(3)中所述CaCl2水溶液的浓度为100mM，成胶时间为60s。经本发明人研究发现，在上述成胶时间下，所获得的营养胶囊的厚度最佳，立体球形胶囊的稳定性最好，且降解的时间仅需10-13min，更符合临床实际操作的需求。Further, the concentration of the CaCl2 aqueous solution in step (3) is 100 mM, and the gelation time is 60 s. The inventors found that under the above-mentioned gelation time, the thickness of the obtained nutritional capsules is the best, the stability of the three-dimensional spherical capsules is the best, and the degradation time is only 10-13 minutes, which is more in line with the needs of clinical practice. .
进一步的是，步骤(1)至步骤(3)中将所得产物均经过杀菌处理，如可以采用450nm和220nm的滤膜过滤除菌来实现杀菌。Further, the products obtained in step (1) to step (3) are all subjected to sterilizing treatment, for example, 450nm and 220nm filter membranes can be used to filter and sterilize to achieve sterilization.
进一步的是，上述步骤(2)中还包括将角膜组织置于预凝胶溶液中，然后进行步骤(3)的操作，该操作用于将角膜组织如角膜基质透镜包裹在营养胶囊的内部，用于中长期立体保存。Further, the above step (2) also includes placing the corneal tissue in the pre-gel solution, and then performing the operation of step (3), which is used to wrap the corneal tissue such as the corneal stromal lens inside the nutritional capsule, For medium and long-term three-dimensional preservation.
本发明的有益效果如下：The beneficial effects of the present invention are as follows:
(1)本发明将含有海藻酸钠的营养保存液与钙离子快速扩散配位形成立体缓冲胶囊球外壳，并进一步构建得到了一种可维持人体泪液稳态的营养胶囊结构，该营养胶囊具有与人体泪液结构组成相度相似的结构，用于保存角膜基质透镜，起到了很好的立体保存、营养供给、稳定缓冲的作用；经过该营养胶囊保存28天后的角膜基质透镜的透光率、细胞活性和胶原纤维密度均能够很好保持，显著优于现有的角膜保存液，并与新鲜的角膜基质透镜相当，可很好维持角膜组织的特性，可实现中长期保存。(1) The present invention coordinates the nutrient preservation solution containing sodium alginate with the rapid diffusion of calcium ions to form a three-dimensional buffer capsule spherical shell, and further constructs a nutrient capsule structure that can maintain the steady state of human tears. The nutrient capsule has The structure similar to the structure of human tears is used to preserve the corneal stroma lens, which plays a good role in three-dimensional preservation, nutrient supply, and stable buffering; the light transmittance, light transmittance, and Cell activity and collagen fiber density can be well maintained, significantly better than existing corneal preservation solutions, and comparable to fresh corneal stroma lenses, which can well maintain the characteristics of corneal tissue and achieve medium and long-term preservation.
(2)将经本发明的营养胶囊保存的角膜基质透镜用于人眼异体透镜移植治疗复杂性屈光不正或角膜疾病，其安全有效，植入的透镜保持高度透明，无移位和排斥现象，术后3月70％术眼的最佳矫正视力较术前增加至少两行。该营养胶囊不仅能为角膜组织的立体保存、运输及再利用提供一种简单、安全、可控的策略，也可为其他活体组织或器官保存提供新的思路。(2) The corneal stromal lens preserved by the nutritional capsule of the present invention is used for human eye allogeneic lens transplantation to treat complex ametropia or corneal disease, which is safe and effective, and the implanted lens remains highly transparent without displacement and rejection , the best corrected visual acuity of 70% of the operated eyes at 3 months after operation increased by at least two lines compared with preoperative. The nutrition capsule can not only provide a simple, safe and controllable strategy for the three-dimensional preservation, transportation and reuse of corneal tissue, but also provide a new idea for the preservation of other living tissues or organs.
附图说明Description of drawings
图1为与人泪液结构高度相似的立体营养胶囊的制备与表征；a)立体营养胶囊包被角膜组织的制备与设计：1.将角膜基质透镜直接置于200μLA溶液中；2.用1mL移液枪吸出含微孔的溶液A；3.用1mL移液枪将含有角膜基质透镜的溶液A移入3mL溶液B中；4.形成水凝胶营养胶囊包埋角膜组织；5.加入含有营养液的培养液3mL，营养胶囊4℃保存；b)营养胶囊包埋人角膜基质透镜的实物摄影；c)不同浓度海藻酸钠凝胶溶液的粘度和球形效率；d)不同凝胶时间营养胶囊的厚度；e)营养胶囊的初始降解时间和完全降解时间；f)营养胶囊完全降解过程的摄影；g)共聚焦显微镜观察硫酸软骨素在透镜上的吸附情况；h)营养胶囊包埋人角膜基质透镜切面图；i)扫描电子显微镜(SEM)观察到的囊壳层结构和物理外观；j)营养胶囊培养5天后释放细胞的活/死染色图像；k)营养胶囊组与空白对照组培养3d和5d细胞CCK-8存活率；Con：空白对照组；NC：营养胶囊组。Figure 1 shows the preparation and characterization of three-dimensional nutritional capsules highly similar to human tears; a) The preparation and design of three-dimensional nutritional capsules coated with corneal tissue: 1. Put the corneal stromal lens directly in 200μLA solution; 2. Use 1mL pipette 3. Use a 1mL pipette gun to transfer solution A containing corneal stromal lenses into 3mL solution B; 4. Form hydrogel nutrient capsules to embed corneal tissue; 5. Add nutrient solution 3mL of culture medium, and the nutrition capsules were stored at 4°C; b) Physical photography of human corneal stroma lenses embedded in nutrition capsules; c) Viscosity and spherical efficiency of sodium alginate gel solutions with different concentrations; d) Nutrient capsules with different gelation times Thickness; e) The initial degradation time and complete degradation time of the nutrition capsule; f) Photography of the complete degradation process of the nutrition capsule; g) Observation of the adsorption of chondroitin sulfate on the lens by confocal microscope; h) Human corneal stroma embedded in the nutrition capsule Lens section view; i) capsule shell structure and physical appearance observed by scanning electron microscope (SEM); j) live/dead staining images of cells released from nutrient capsules cultured for 5 days; k) nutrient capsules group and blank control group cultured for 3 days and 5d cell CCK-8 survival rate; Con: blank control group; NC: nutrition capsule group.
图2中a分图为培养液中的角膜基质透镜(左)和不同厚度、不同Ca2+成胶时间的营养胶囊(右)，其中成胶300s后的营养胶囊为实心结构；b分图为保存3天和7天不同成胶时间的营养胶囊保存的角膜透镜的活/死染色；c分图为保存3天和7天不同成胶时间的营养胶囊保存角膜透镜的活/死染色定量分析。Part a of Fig. 2 shows the corneal stroma lens (left) in the culture medium and the nutritional capsules (right) with different thicknesses and different Ca2+ gelation times, and the nutritional capsules after 300s of gelation are solid structures; part b Live/dead staining of corneal lenses stored in nutritional capsules with different gelation times for 3 days and 7 days; sub-graph c is quantification of live/dead staining for corneal lenses stored in nutritional capsules with different gelation times for 3 days and 7 days analyze.
图3为不同方法保存的角膜透镜TUNEL染色和DAPI染色细胞核结果。Figure 3 shows the results of TUNEL staining and DAPI staining nuclei of corneal lenses preserved by different methods.
图4为各处理组TUNEL阳性细胞的平均百分比。Figure 4 shows the average percentage of TUNEL positive cells in each treatment group.
图5为各处理组角膜基质透镜的活/死染色，活细胞(绿色)和死细胞(红色)分别与Calcein-AM(活细胞)和同源二聚体-1(死细胞)溶液孵育。Figure 5 is the live/dead staining of corneal stromal lenses in each treatment group, live cells (green) and dead cells (red) were incubated with Calcein-AM (live cells) and homodimer-1 (dead cells) solutions, respectively.
图6为各处理组中活细胞和死细胞的平均百分比；Con：空白对照组；NC：营养胶囊组，数据以平均标准差表示，*P<0.05，****P<0.0001。Figure 6 shows the average percentage of living cells and dead cells in each treatment group; Con: blank control group; NC: nutrition capsule group, the data are expressed as mean standard deviation, *P<0.05, ****P<0.0001.
图7为营养胶囊保存的角膜基质透镜的组织结构和透光率；a)HE染色，b)Masson染色，c)VG染色，显示对照组和实验组在不同保存时间下角膜透镜的组织学变化；d)各组胶原纤维横断面透射电镜观察；e)各组透镜的平均透过率；f)保存7d、14d、28d时对照组与实验组胶原纤维密度比较；Con：空白对照组；NC：营养胶囊组，数据以平均标准差表示，*P<0.05，****P<0.0001。Figure 7 shows the histological structure and light transmittance of corneal stroma lenses preserved in nutritional capsules; a) HE staining, b) Masson staining, and c) VG staining, showing the histological changes of corneal lenses in the control group and experimental group at different storage times ; d) Transmission electron microscope observation of the cross-section of collagen fibers in each group; e) Average transmittance of lenses in each group; f) Comparison of collagen fiber density between the control group and the experimental group when stored for 7d, 14d, and 28d; Con: blank control group; NC : Nutrient capsule group, the data are represented by mean standard deviation, *P<0.05, ****P<0.0001.
图8角膜疾病患者进行营养胶囊保存的角膜透镜移植术前、术后1个月裂隙灯与眼前节光学相干断层扫描(AS-OCT)比较。Figure 8 Comparison of slit-lamp and anterior segment optical coherence tomography (AS-OCT) before and 1 month after corneal lens transplantation in patients with corneal diseases who underwent nutrient capsule preservation.
图9中a为人体泪液的结构组成示意图，b为营养胶囊的结构组成示意图。In Figure 9, a is a schematic diagram of the structure and composition of human tears, and b is a schematic diagram of the structure and composition of nutritional capsules.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例对本发明进行具体描述，有必要指出的是，以下实施例仅用于对本发明进行解释和说明，并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整，仍属于本发明的保护范围。In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below in conjunction with the examples. It must be pointed out that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the present invention . Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.
实施例1Example 1
本实施例提供了一种营养胶囊的制备方法，具体如下：The present embodiment provides a kind of preparation method of nutrition capsule, specifically as follows:
1、初始溶液的配置1. Configuration of the initial solution
首先，以角膜基础培养基DMEM/F-12(其成分为：45％DMEM和45％F-12)作为母液，并加入硫酸软骨素、双抗和海藻糖使其终浓度分别为2.5wt％、1wt％和0.5wt％，待上述溶液溶解完全后，加入海藻酸钠，使海藻酸钠的终浓度为1wt％，上述操作均在无菌条件下进行，待完全溶解后，溶液分别经450nm和220nm的滤膜过滤除菌，得到A组分溶液。First, the corneal basal medium DMEM/F-12 (its composition is: 45% DMEM and 45% F-12) was used as the mother solution, and chondroitin sulfate, double antibody and trehalose were added to make the final concentrations respectively 2.5wt% , 1wt% and 0.5wt%. After the above solution is completely dissolved, add sodium alginate to make the final concentration of sodium alginate 1wt%. The above operations are all carried out under sterile conditions. And 220nm membrane filtration sterilization, obtain A component solution.
随后，用无菌水配置100mM CaCl2水溶液(B组分溶液)和100mM柠檬酸钠水溶液(C组分溶液)，溶解后经450nm和220nm滤膜过滤除菌。Subsequently, 100 mM CaCl2 aqueous solution (B component solution) and 100 mM sodium citrate aqueous solution (C component solution) were prepared with sterile water, and after dissolution, they were filtered through 450 nm and 220 nm filter membranes to sterilize.
2、营养胶囊的制备2. Preparation of Nutritional Capsules
移取1mLA组分溶液滴加入3mL B组分溶液中，静置成胶60s形成水凝胶胶囊，随后移去多余B组分，并用3mLPBS快速洗涤3次以除去过量B组分，得到营养胶囊。Pipette 1mL component A solution into 3mL component B solution dropwise, let it stand for 60s to form a hydrogel capsule, then remove excess component B, and quickly wash with 3mL PBS 3 times to remove excess component B to obtain nutritional capsules .
以加入3mL含有营养物质的溶液作为培养基，于4℃保存备用。Add 3 mL of a solution containing nutrients as a medium, and store it at 4°C for later use.
实施例2Example 2
按照实施例1的方法制备营养胶囊，其中将硫酸软骨素、双抗、海藻糖的浓度依次调整为0.1％、0.5％、0.2％，同时添加0.01％的抗坏血酸钠，待溶解后向其中添加0.5％的海藻酸钠，其它方法不变。Nutritional capsules were prepared according to the method of Example 1, wherein the concentrations of chondroitin sulfate, double antibody, and trehalose were adjusted to 0.1%, 0.5%, and 0.2% in sequence, and 0.01% sodium ascorbate was added at the same time, and 0.5% sodium ascorbate was added to it after dissolution. % sodium alginate, other methods remain unchanged.
实施例3Example 3
按照实施例1的方法制备营养胶囊，其中将硫酸软骨素、双抗、海藻糖的浓度依次调整为3％、2％、1％，同时添加0.3％的抗坏血酸钠，待溶解后向其中添加3％的海藻酸钠，其它方法不变。Nutritional capsules were prepared according to the method of Example 1, wherein the concentrations of chondroitin sulfate, double antibody, and trehalose were adjusted to 3%, 2%, and 1% in sequence, and 0.3% sodium ascorbate was added at the same time, and 3 % sodium alginate, other methods remain unchanged.
对比例1Comparative example 1
专利文献CN 104046587 A中报道了一种含有硫酸软骨素的海藻酸钙微胶珠(其为实心结构)，因此本对比例摸索了将营养胶囊制备成实心结构用于保存角膜基质透镜，实心营养胶囊的具体制备方法如下：参照实施例1的制备方法，将包含角膜基质透镜的预凝胶溶液加入钙离子溶液中时，延长成胶时间到300s时胶囊成为实心。The patent document CN 104046587 A reports a calcium alginate microgel beads containing chondroitin sulfate (it has a solid structure), so this comparative example explores the preparation of nutritional capsules into a solid structure for preserving the corneal stroma lens, solid nutrition The specific preparation method of the capsule is as follows: referring to the preparation method of Example 1, when the pregel solution containing the corneal stromal lens is added to the calcium ion solution, the capsule becomes solid when the gelation time is extended to 300s.
将成胶300s获得的实心营养胶囊用于角膜基质透镜的保存，考查其中长期保存效果。The solid nutrient capsule obtained by gelling for 300s was used to preserve the corneal stroma lens, and the long-term preservation effect was examined.
对比例2Comparative example 2
以专利文献CN 113907066A中实施例4记载的保存液(以1000mL保存液计：硫酸软骨素38mg、HEPES缓冲液4.5g、抗生素妥布霉素40mg、细胞营养成分谷氨酰胺200g，余量为含有酸碱调节剂的MEM培养液，pH值7.6)用于角膜基质透镜的中长期保存，考查该保存液保存14天和28天后对角膜基质透镜的纤维结构和透明度、以及角膜基质细胞活性的影响。With the preservation solution described in Example 4 in the patent document CN 113907066A (in terms of 1000mL preservation solution: chondroitin sulfate 38mg, HEPES buffer 4.5g, antibiotic tobramycin 40mg, cell nutrient glutamine 200g, the balance is containing MEM culture solution with acid-base regulator, pH value 7.6) is used for medium and long-term preservation of corneal stromal lenses, and the effects of the preservation solution on the fiber structure and transparency of corneal stromal lenses and the activity of corneal stromal cells after 14 days and 28 days of storage are investigated .
对比例3Comparative example 3
以专利文献CN 110384088 A中实施例采用的保存方法(硫酸软骨素-透明质酸钠粘弹剂，由3％的透明质酸钠和4％的硫酸软骨素钠和生理缓冲平衡盐组成的无色透明凝胶状溶液)用于角膜基质透镜的中长期保存，考查该保存方法保存14天和28天后对角膜基质透镜的纤维结构和透明度、以及角膜基质细胞活性的影响。With the preservation method (chondroitin sulfate-sodium hyaluronate viscoelastic agent, composed of 3% sodium hyaluronate and 4% chondroitin sulfate sodium and physiological buffer balance salt) Color transparent gel-like solution) is used for medium and long-term preservation of corneal stromal lenses, and the effects of this preservation method on the fiber structure and transparency of corneal stromal lenses and the activity of corneal stromal cells after 14 days and 28 days of storage are investigated.
对比例4Comparative example 4
以专利文献CN 104094925 B中实施例记载的保存液(以序号1的组分为例，含甘油200g/L、透明质酸5g/L、硫酸软骨素25g/L、抗氧化剂N-乙酰半胱氨酸5g/L、磷酸盐缓冲液30mmol/L)用于角膜基质透镜的中长期保存，考查该保存方法保存14天和28天后对角膜基质透镜的纤维结构和透明度，以及角膜基质细胞活性的影响。Take the preservation solution described in the examples of patent document CN 104094925 B (taking the component of serial number 1 as an example, containing glycerin 200g/L, hyaluronic acid 5g/L, chondroitin sulfate 25g/L, antioxidant N-acetylcysteine Amino acid 5g/L, phosphate buffer saline 30mmol/L) is used for medium and long-term preservation of corneal stromal lenses, and the effects of this preservation method on the fiber structure and transparency of corneal stromal lenses and the activity of corneal stromal cells after 14 days and 28 days of storage are investigated. Influence.
对比例5Comparative example 5
以专利文献CN 109699631 A中实施例记载的保存液配方(2.0g低熔点琼脂糖和0.1g葡聚糖(分子量40kDa)分散于80ml浓度为0.2mol/L的PBS缓冲液中，加热不断搅拌直至完全溶胀，加入0.1g甘油，搅拌均匀，PBS缓冲液稀释至100ml，使最终pH为7.2～7.4)用于角膜基质透镜的中长期保存，考查该保存方法保存14天和28天后对角膜基质透镜的纤维结构和透明度，以及角膜基质细胞活性的影响。With the preservation solution formula (2.0g low-melting point agarose and 0.1g dextran (molecular weight 40kDa) that is recorded in the example record in patent document CN 109699631 A, be dispersed in the PBS buffer solution that 80ml concentration is 0.2mol/L, heat and stir constantly until Completely swell, add 0.1g glycerin, stir evenly, dilute to 100ml with PBS buffer, so that the final pH is 7.2~7.4) It is used for medium and long-term storage of corneal stroma lens. Effects of fiber structure and transparency, and corneal stromal cell activity.
阳性对照例1Positive Control Example 1
以甘油作为保存液，用于角膜基质透镜的中长期保存，作为阳性对照组一。Glycerin was used as a preservation solution for medium and long-term preservation of corneal stromal lenses, and it was used as positive control group 1.
阳性对照例2Positive control example 2
以Optisol-GS(Bausch&Lomb Inc.，Bridgewater，NJ，USA)作为保存液，用于角膜基质透镜的中长期保存，作为阳性对照组二。Optisol-GS (Bausch & Lomb Inc., Bridgewater, NJ, USA) was used as a preservation solution for medium and long-term storage of corneal stromal lenses, as the second positive control group.
阳性对照例3Positive control example 3
以DMEM培养基(Gibco，Thermo Fisher Scientific，USA)作为保存液，用于角膜基质透镜的中长期保存，作为阳性对照组三。DMEM medium (Gibco, Thermo Fisher Scientific, USA) was used as a preservation solution for mid- and long-term preservation of corneal stromal lenses, as positive control group 3.
实验例1Experimental example 1
对实施例中制备得到的营养胶囊进行物理表征，并考查不同制备条件对营养胶囊的影响Physically characterize the nutritional capsules prepared in the examples, and examine the effects of different preparation conditions on the nutritional capsules
(一)表征方法(1) Representation method
(1)通过流变仪(TA-DHR-2)测定不同浓度海藻酸钠凝胶母液的黏度，并拍照观察不同黏度母液的成球效果。(1) Measure the viscosities of different concentrations of sodium alginate gel mother liquors by rheometer (TA-DHR-2), and take pictures to observe the balling effect of mother liquors with different viscosities.
(2)在B组分中加入罗丹明，用激光共聚焦检测不同成胶时间胶囊的形成厚度。(2) Rhodamine was added to component B, and laser confocal was used to detect the formation thickness of different gelation time capsules.
(3)通过体外拍照观察和显微镜实时监测营养胶囊初始降解时间和完全降解时间，并通过GelMA光敏凝胶包埋切割和SEM扫描电镜观察营养胶囊的立体结构和物理形貌。此外，利用Rhodamine B isothiocyanate(罗丹明B异硫氰酸酯)标记的硫酸软骨素(Chs-Rho)与透镜共孵育1h，并用生理盐水洗涤2h以洗去未吸附的Chs-Rho，用激光共聚焦(蔡司，德国)镜头进行观察。(3) The initial degradation time and complete degradation time of the nutritional capsules were monitored in real time by in vitro photographic observation and microscopy, and the three-dimensional structure and physical morphology of the nutritional capsules were observed by GelMA photosensitive gel embedding and cutting and SEM scanning electron microscopy. In addition, Rhodamine B isothiocyanate (Rhodamine B isothiocyanate) labeled chondroitin sulfate (Chs-Rho) was co-incubated with the lens for 1 h, and washed with normal saline for 2 h to wash away the unadsorbed Chs-Rho, and laser co-incubated A focusing (Zeiss, Germany) lens was used for observation.
(二)表征结果(2) Characterization results
1、按照实施例1的方法制备营养胶囊，考查了海藻酸钠的浓度分别为0.5％、1％、1.5％和2％对营养胶囊立体成球性的影响，结果显示：1％浓度的海藻酸钠溶液比0.5％、1.5％和2％浓度成球更加均匀；虽然从预凝胶溶液的黏度发现2％和1.5％预凝胶溶液的起始黏度较高，分别达到了4.27Pa·S和1.17Pa·S，易于形成泪滴状胶囊，而0.5％预凝胶溶液黏度较低只有0.059Pa·S，形成的胶囊支撑性较差，但1％浓度的预凝胶溶液的成球均匀性更佳。因此，后续实验均选用1％浓度的海藻酸钠作为预凝胶溶液。1. Prepare nutritional capsules according to the method in Example 1, and examine the effects of sodium alginate concentrations of 0.5%, 1%, 1.5% and 2% on the three-dimensional spherical properties of nutritional capsules. The results show that: 1% concentration of seaweed Sodium acid solution is more uniform than 0.5%, 1.5% and 2% concentration; although it is found from the viscosity of pregel solution that the initial viscosity of 2% and 1.5% pregel solution is higher, reaching 4.27Pa·S respectively and 1.17Pa·S, it is easy to form teardrop-shaped capsules, while the viscosity of 0.5% pregel solution is only 0.059Pa·S, and the support of the formed capsule is poor, but the sphere of 1% pregel solution is uniform Sex is better. Therefore, 1% sodium alginate was used as the pregel solution in subsequent experiments.
2、在钙离子溶液中加入罗丹明并用共聚焦显微镜观察钙离子渗透时间与形成胶囊厚度的关系。从图1中d分图可以看出，当预凝胶溶液接触钙离子溶液10s时就形成了约224.26μm的膜厚度，并随着时间的延长膜厚度继续增加，当成胶60s时膜厚度达到了303.44μm。2. Add rhodamine to the calcium ion solution and observe the relationship between the calcium ion penetration time and the thickness of the formed capsules with a confocal microscope. It can be seen from the sub-graph d in Figure 1 that when the pre-gel solution contacts the calcium ion solution for 10 seconds, a film thickness of about 224.26 μm is formed, and the film thickness continues to increase as time goes on. When the gel is formed for 60 seconds, the film thickness reaches 303.44μm.
进一步考查了营养胶囊在100mM柠檬酸钠溶液中的初始降解时间和最终降解时间，并通过体外大体照片和显微镜照片检测了成胶60s营养胶囊的降解全过程。从图1中e分图和f分图可以看出，在7min时营养胶囊表面开始发生破裂，9min中胶囊中溶液缓慢渗出，13min胶囊基本降解。相比较其它交联时间，交联60s凝胶更加稳定，且降解操作时间更加符合临床实际操作。The initial degradation time and final degradation time of the nutritional capsules in 100mM sodium citrate solution were further examined, and the whole degradation process of the gelatinized 60s nutritional capsules was detected by in vitro gross photos and microscopic photos. It can be seen from the sub-graphs e and f in Figure 1 that the surface of the nutritional capsule began to crack at 7 minutes, the solution in the capsule slowly leaked out at 9 minutes, and the capsule was basically degraded at 13 minutes. Compared with other cross-linking times, the cross-linked 60s gel is more stable, and the degradation operation time is more in line with clinical practice.
3、为了更加直观观察胶囊结构，将营养胶囊包埋在20％光敏GelMA溶液中，通过10mW/cm2365 nm UV光源交联2min使得凝胶溶液完全固化，并用手术刀片将其分隔开，如图1中h分图所示，可以清晰地看到微球的壳层结构，而且角膜透镜仍可保持初始结构。此外，通过SEM观察胶囊的内壁和胶囊结构，从图1中i分图可以看出胶囊内壁呈现凹凸褶皱结构，壳层厚度约为24μm。3. In order to observe the capsule structure more intuitively, the nutritional capsules were embedded in 20% photosensitive GelMA solution, cross-linked by 10mW/cm2 365 nm UV light source for 2min to completely solidify the gel solution, and separated with a scalpel, As shown in sub-graph h of Figure 1, the shell structure of the microspheres can be clearly seen, and the corneal lens can still maintain the original structure. In addition, the inner wall and capsule structure of the capsule were observed by SEM. It can be seen from the sub-graph i in Figure 1 that the inner wall of the capsule presents a concave-convex wrinkled structure, and the thickness of the shell layer is about 24 μm.
4、为了验证硫酸软骨素对透镜的静电吸附作用，将透镜浸没在罗丹明(Rho)标记的硫酸软骨素Chs-Rho中，从图1中g分图发现，Chs(硫酸软骨素)对透镜有明显吸附作用，而且通过3D图可以清晰看出Chs可以渗透整个透镜。4. In order to verify the electrostatic adsorption of chondroitin sulfate to the lens, the lens was immersed in rhodamine (Rho)-labeled chondroitin sulfate Chs-Rho. It was found from the g subgraph in Figure 1 that Chs (chondroitin sulfate) had a strong effect on the lens. It has obvious adsorption effect, and it can be clearly seen from the 3D image that Chs can penetrate the entire lens.
5、为了验证营养胶囊的生物相容性，将人角膜基质细胞(HCSC)包裹在营养胶囊中，保存一周后释放于培养基中进行体外培养3天和5天，通过活死染色和CCK8实验来检测营养胶囊保存对细胞的影响。图1中j分图活死染色结果显示，绝大多数细胞在这3天和5天的培养过程中存活，组间没有统计学差异。与活死染色的结果相一致，CCK8实验结果显示，在体外培养5天过程中，各组细胞均持续增殖，实验组第3天和第5天细胞生存率与对照组差异无统计学意义(P＝0.59)和(P＝0.08)(图1中k分图)。这些结果证明，营养胶囊具有良好的生物相容性，能够为角膜组织透镜提供一个良好的立体储存环境。5. In order to verify the biocompatibility of the nutritional capsules, the human corneal stromal cells (HCSC) were wrapped in the nutritional capsules, stored in the medium for one week and then released in the culture medium for 3 days and 5 days in vitro, through the live-death staining and CCK8 experiments To detect the effect of nutrient capsule preservation on cells. The live-dead staining results of sub-graph j in Figure 1 showed that most of the cells survived during the 3-day and 5-day culture, and there was no statistical difference between the groups. Consistent with the results of live-dead staining, the results of CCK8 experiment showed that during the 5-day culture in vitro, the cells in each group continued to proliferate, and the cell survival rate of the experimental group on the 3rd and 5th day was not significantly different from that of the control group ( P=0.59) and (P=0.08) (k sub-plot in Figure 1). These results prove that the nutritional capsule has good biocompatibility and can provide a good three-dimensional storage environment for the corneal tissue lens.
实验例2Experimental example 2
以实施例1记载的方法制备营养胶囊，并设置不同成胶时间(30s、60s、120s、180s、300s)，考查不同成胶时间对营养胶囊结构的影响，以及对角膜基质细胞活性保存效果的影响。Prepare nutritional capsules with the method described in Example 1, and set different gelation times (30s, 60s, 120s, 180s, 300s), examine the impact of different gelation times on the structure of nutrition capsules, and the preservation effect on the activity of corneal stromal cells Influence.
(一)实验方法：将SMILE手术提取的角膜基质透镜置于200μLA溶液中，然后用1ml移液枪将含有角膜基质透镜的溶液A移入3ml溶液B中，形成水凝胶营养胶囊。观察不同成胶时间对人角膜基质细胞活性的影响。将角膜基质透镜包裹在不同成胶时间(30s、60s、120s、180s、300s)的营养胶囊中，去除多余的溶液B，用3mLPBS洗涤水凝胶3次。加入含有营养成分的角膜培养基3mL，将营养胶囊储存在4℃。用Live/Dead试剂盒(日本DojindoLaboratorise)染色，并在第3天和第7天使用荧光显微镜(德国蔡司)拍照。将成胶不同时间的营养胶囊置于培养液中，于4℃条件下分别保存3天和7天后，进行Live/Dead染色。实验结果见图2。(1) Experimental method: Put the corneal stroma lens extracted by SMILE into 200μLA solution, and then use a 1ml pipette gun to transfer solution A containing the corneal stroma lens into 3ml solution B to form a hydrogel nutritional capsule. To observe the effect of different gelation time on the activity of human corneal stromal cells. The corneal stromal lens was wrapped in nutritional capsules with different gelation times (30s, 60s, 120s, 180s, 300s), excess solution B was removed, and the hydrogel was washed 3 times with 3 mL of LPBS. Add 3 mL of corneal medium containing nutrients, and store the nutrition capsules at 4 °C. Stained with Live/Dead kit (Dojindo Laboratorise, Japan), and photographed using a fluorescence microscope (Zeiss, Germany) on day 3 and day 7. The nutrient capsules that had been gelled for different times were placed in the culture medium, stored at 4°C for 3 days and 7 days, respectively, and then Live/Dead staining was performed. The experimental results are shown in Figure 2.
(二)实验结果：从图2的结果可以看出，成胶时间为60s的营养胶囊中的角膜基质透镜的活细胞数量明显多于其他成胶时间营养胶囊保存的角膜基质透镜活细胞数量，且该方法保存的角膜基质透镜特性显著优于实心胶囊保存的角膜基质透镜。分析主要原因：1)交联时间过长，钙离子浓度越高，对细胞的毒性越大；2)交联时间越长，胶囊外壳收缩越明显，交联收缩过程中的物理拉力是影响细胞活性和角膜透光率的主要原因；3)随着交联时间的增加，降解时间也相应增加，导致角膜基质透镜内活细胞数量逐渐减少，角膜胶原结构容易被破坏。(2) Experimental results: as can be seen from the results in Fig. 2, the number of viable cells of the corneal stroma lens in the nutrient capsules with gelation time of 60s is obviously more than that of the corneal stroma lens live cells preserved in other gelation time nutrition capsules, Moreover, the characteristics of the corneal stroma lens preserved by this method are significantly better than those of the corneal stroma lens preserved by the solid capsule. The main reasons for the analysis: 1) If the cross-linking time is too long, the higher the calcium ion concentration, the greater the toxicity to the cells; 2) The longer the cross-linking time, the more obvious the shrinkage of the capsule shell, and the physical tension in the process of cross-linking shrinkage is the most important factor affecting the cells. The main reason is activity and corneal light transmittance; 3) With the increase of cross-linking time, the degradation time also increases accordingly, resulting in the gradual decrease of the number of living cells in the corneal stroma lens, and the corneal collagen structure is easily destroyed.
实验例3Experimental example 3
本实验例考查营养胶囊以及不同保存方法用于中长期保存角膜基质透镜后的保存效果。This experimental example examines the preservation effect of nutritional capsules and different preservation methods for medium and long-term preservation of corneal stroma lenses.
(一)实验方法(1) Experimental method
1、人角膜基质细胞培养1. Human corneal stromal cell culture
使用人角膜基质细胞(HCSC)进行实验，HCSC在DMEM/F-12培养基中与10％的胎牛血清以及100U/mL青霉素和100U/mL链霉素的抗生素混合物(双抗)中生长。其中，HCSC来源于中国复旦大学眼科耳鼻喉科医院，DMEM/F-12、胎牛血清、青霉素和链霉素均来源于Gibco，Thermo Fisher Scientific，USA。Human corneal stromal cells (HCSC) were used for experiments, and HCSC were grown in DMEM/F-12 medium with 10% fetal bovine serum and an antibiotic mixture (double antibody) of 100 U/mL penicillin and 100 U/mL streptomycin. Among them, HCSC was obtained from Otolaryngology Hospital of Fudan University, China, and DMEM/F-12, fetal bovine serum, penicillin and streptomycin were all obtained from Gibco, Thermo Fisher Scientific, USA.
2、营养胶囊微生物相容性测试2. Microbial compatibility test of nutritional capsules
通过1mL注射器将含细胞悬液(5×104细胞浓度)的海藻酸钠凝胶预溶液缓慢滴入100mM氯化钙溶液，交联1min形成细胞凝胶微珠，用DMEM/F-12基础培养基洗涤，并在1mL新鲜培养基中重悬。将装有海藻酸钙包覆细胞的小瓶于4℃冷藏7天，存储7天后，用PBS清洗包裹细胞的海藻酸钙珠，然后加入到C组分溶液中使其溶解，溶解10min后，以1000转/分钟离心5分钟，沉积的细胞用培养基重悬。此外，将不含细胞的海藻酸钙微珠储存在含有营养物质的完全培养基中，保存7天，取出海藻酸钙珠，然后用浸泡液培养细胞。将海藻酸钙包裹的细胞释放后用完全培养基培养3天和5天或用海藻酸钙浸泡液培养3天和5天的细胞，每孔共计3×104个细胞接种于96孔板；进行CCK8检测(碧云天生物科技，中国)，用酶标仪(Tecan，Switzerland)测量其光密度值(OD)，并用Live/Dead试剂盒(DojindoLaboratorise，日本)对不同处理的细胞进行染色，使用荧光显微镜(蔡司，德国)观察和拍摄。Slowly drop the sodium alginate gel pre-solution containing cell suspension (5×104 cell concentration) into 100mM calcium chloride solution through a 1mL syringe, cross-link for 1min to form cell gel microbeads, and use DMEM/F-12 base Medium was washed and resuspended in 1 mL of fresh medium. Refrigerate the vial containing the calcium alginate-coated cells at 4°C for 7 days. After 7 days of storage, wash the calcium alginate beads that coated the cells with PBS, and then add them to the solution of component C to dissolve them. After dissolving for 10 minutes, use Centrifuge at 1000 rpm for 5 minutes, and resuspend the deposited cells with culture medium. In addition, the calcium alginate microbeads without cells were stored in complete medium containing nutrients for 7 days, the calcium alginate beads were removed, and then the cells were cultured with the soaking solution. After releasing the calcium alginate-coated cells, culture them with complete medium for 3 days and 5 days or with calcium alginate soaking solution for 3 days and 5 days, and inoculate a total of 3×104 cells per well in a 96-well plate; Carry out CCK8 detection (Beiyuntian Biotechnology, China), with a microplate reader (Tecan, Switzerland) to measure the optical density value (OD), and the cells with different treatments were stained with the Live/Dead kit (Dojindo Laboratorise, Japan), and observed and photographed using a fluorescence microscope (Zeiss, Germany).
3、营养胶囊包裹角膜基质透镜的生物特性研究3. Study on the biological characteristics of corneal stroma lens wrapped in nutritional capsules
(1)角膜基质透镜不同保存方法的比较(1) Comparison of different preservation methods for corneal stromal lenses
采用本发明实施例中获得的营养胶囊保存角膜基质透镜，并与对比例和阳性对照例中的方法进行比较，考查各种保存方法对角膜基质透镜中长期保存的效果。具体方法如下：The nutrient capsule obtained in the embodiment of the present invention was used to preserve the corneal stroma lens, and compared with the methods in the comparative example and the positive control example, the effect of various preservation methods on the medium and long-term preservation of the corneal stroma lens was investigated. The specific method is as follows:
实验分为不同组进行，阳性对照组一：99％无水甘油(阳性对比例1)；阳性对照组二：Optisol-GS(阳性对比例2)；阳性对照组三：DMEM培养基(阳性对比例3)；实验组(以实施例1中获得的营养胶囊为例)。取出需要保存的角膜基质透镜在磷酸盐缓冲液(PBS)中洗涤两次，随机分为不同组添加到上述保存液中进行保存，放置于4℃条件下分别保存7天、14天和28天。Experiment is divided into different groups to carry out, positive control group one: 99% anhydrous glycerol (positive contrast example 1); Positive control group two: Optisol-GS (positive contrast example 2); Positive control group three: DMEM medium (positive contrast example 2); Ratio 3); Experimental group (taking the nutritional capsule obtained in Example 1 as an example). Take out the corneal stromal lenses that need to be preserved, wash them twice in phosphate buffered saline (PBS), divide them randomly into different groups, add them to the above-mentioned preservation solution for preservation, and store them at 4°C for 7 days, 14 days and 28 days respectively .
以新鲜透镜(术后取出1小时内)作为空白对照组，与这些保存的角膜组织透镜进行比较。并分别与对比例1～5的方案进行比较。Fresh lenses (taken out within 1 hour after surgery) were used as a blank control group to compare with these preserved corneal tissue lenses. And compare with the schemes of Comparative Examples 1-5 respectively.
(2)角膜基质细胞活性检测(2) Detection of corneal stromal cell activity
检测不同保存条件下各组角膜组织透镜的凋亡细胞，采用TUNEL法，原位细胞死亡检测试剂盒(Roche Applied Science，Indianapolis，IN)根据说明书使用。用Live/Dead试剂盒(日本Dojindo Laboratories)对透镜进行染色，使用荧光显微镜(蔡司，德国)观察和拍摄透镜。The apoptotic cells of each group of corneal tissue lenses under different storage conditions were detected by the TUNEL method, and the in situ cell death detection kit (Roche Applied Science, Indianapolis, IN) was used according to the instructions. The lens was stained with the Live/Dead kit (Dojindo Laboratories, Japan), and the lens was observed and photographed using a fluorescence microscope (Zeiss, Germany).
(3)透光率和显微结构观察(3) Light transmittance and microstructure observation
各组透镜随后置于96孔板中，并用三角海绵签吸干周围的液体。吸光度测量使用酶标仪(Tecan，瑞士)。吸光度(A)在可见光波长下测量，范围为380nm至780nm，每间隔10nm取一个检测点，连续多点检测样本的吸光度，重复测量3次，记录数值，选取空白孔板作为对照，透过率(T)计算公式如下：T＝10-A。Each set of lenses was then placed in a 96-well plate, and the surrounding liquid was blotted with a triangular sponge swab. Absorbance was measured using a microplate reader (Tecan, Switzerland). The absorbance (A) is measured under the wavelength of visible light, the range is 380nm to 780nm, a detection point is taken at an interval of 10nm, the absorbance of the sample is detected at multiple points continuously, the measurement is repeated 3 times, and the value is recorded. A blank orifice plate is selected as a control. (T) The calculation formula is as follows: T=10-A .
取出新鲜透镜或保存的透镜，然后迅速切成小段(1mm3)，这些切片固定在2.5％戊二醛2小时，样品经熏蒸后，用0.1M四氧化锇燃烧0.5-2h，使其完全碳化。用100％丙酮漂洗2次5min后，与100％丙酮和环氧树脂溶液混合，室温静置15min。样品被放置在烤箱38℃2-3h，随后样本表面覆盖组织面丙酮，加环氧树脂和100％丙酮混合液，置入38℃烘箱内2-3小时，尽量除去混合浸透液置换成纯环氧树脂，并置于38℃烘箱内1小时。超薄切片(70nm)使用徕卡EM-UC7(徕卡生物系统，柏林，德国)进行切割，标本用3％醋酸铀酰和柠檬酸铅双重染色，随后在120kV的Tecnai G2 Spirit BioTWIN电子显微镜(FEI，Portland，US)下观察和成像。Take out the fresh lens or the preserved lens, and then quickly cut into small sections (1mm3 ), these sections are fixed in 2.5% glutaraldehyde for 2 hours, after the sample is fumigated, burn with 0.1M osmium tetroxide for 0.5-2h to make it completely carbonized . Rinse twice with 100% acetone for 5 minutes, mix with 100% acetone and epoxy resin solution, and let it stand at room temperature for 15 minutes. The sample is placed in an oven at 38°C for 2-3 hours, then the surface of the sample is covered with acetone on the tissue surface, a mixture of epoxy resin and 100% acetone is added, and it is placed in an oven at 38°C for 2-3 hours, and the mixed soaking liquid is removed as much as possible and replaced with a pure ring oxygen resin and placed in a 38°C oven for 1 hour. Ultra-thin sections (70nm) were cut using Leica EM-UC7 (Leica Biosystems, Berlin, Germany), and the specimens were double-stained with 3% uranyl acetate and lead citrate, followed by a Tecnai G2 Spirit BioTWIN electron microscope at 120kV (FEI, Portland, US) for observation and imaging.
(4)组织学及免疫组织化学检测(4) Histological and immunohistochemical tests
角膜透镜切片HE染色进行组织病理学光镜下观察，Van Gieson(VG)染色及Masson染色进行胶原结构观察，免疫组织化学检测HLA-DR、HLA-ABC和CD45的表达。Corneal lens sections were stained with HE staining for histopathological light microscope observation, Van Gieson (VG) staining and Masson staining for collagen structure observation, immunohistochemical detection of HLA-DR, HLA-ABC and CD45 expression.
(二)实验结果(2) Experimental results
1、各处理组保存后角膜基质细胞活性检测1. Detection of corneal stromal cell activity after storage in each treatment group
采用TUNEL及活死染色法观察各处理组保存后的透镜中细胞凋亡及存活的数量。实验组、阳性对照组和空白对照组的实验结果如图3-图6所示。TUNEL and live-death staining methods were used to observe the number of apoptosis and survival cells in the preserved lenses of each treatment group. The experimental results of the experimental group, the positive control group and the blank control group are shown in Figures 3-6.
从图3可见，在各处理组保存条件下，7天、14天和28天不同方法保存的各组透镜中均可见Tunel阳性细胞。保存7天后，实验组(NC，营养胶囊组)Tunel阳性细胞率与空白对照组(Con)相比无显著差异，而阳性对照组一(甘油组)(P<0.0001)、阳性对照组二(Optisol组)(P＝0.018)和阳性对照组三(DEME组)(P<0.0001)的Tunel阳性细胞率显著升高。第14天和第28天，各组Tunel阳性细胞率均显著高于空白对照组(P<0.0001)(图4)。It can be seen from Figure 3 that under the storage conditions of each treatment group, Tunel-positive cells can be seen in each group of lenses preserved in different ways for 7 days, 14 days and 28 days. After 7 days of preservation, the rate of Tunel positive cells in the experimental group (NC, nutrition capsule group) had no significant difference compared with the blank control group (Con), while positive control group 1 (glycerol group) (P<0.0001), positive control group 2 ( Optisol group) (P=0.018) and positive control group III (DEME group) (P<0.0001) the rate of Tunel positive cells increased significantly. On day 14 and day 28, the rate of Tunel-positive cells in each group was significantly higher than that in the blank control group (P<0.0001) (Figure 4).
图5和图6显示死活染色的结果支持了TUNEL分析的结果，表明在7-28天不同保存时间内，与其它保存方法相比，营养胶囊保存后的角膜组织透镜存在更多的活细胞。保存7天时，甘油组活细胞较空白对照组显著降低(P<0.0001)，其余组别无显著性差异；保存14天时，DMEM组(P＝0.02)与甘油组(P<0.0001)活细胞较空白对照组显著降低，营养胶囊组及Optisol组与空白对照组相比无显著性差异；保存28天时，仅营养胶囊组与对照组相比无显著性差异，Optisol组(P＝0.03)、DMEM组(P<0.0001)与甘油组(P<0.0001)的活细胞较空白对照组均显著降低。Figure 5 and Figure 6 show that the results of life-and-death staining support the results of TUNEL analysis, indicating that there are more living cells in the corneal tissue lenses stored in nutritional capsules compared with other storage methods during different storage periods of 7-28 days. When stored for 7 days, the viable cells in the glycerol group were significantly lower than those in the blank control group (P<0.0001), and there was no significant difference in the other groups; Compared with the blank control group, the nutritional capsule group and Optisol group had no significant difference; when stored for 28 days, only the nutritional capsule group had no significant difference compared with the control group, the Optisol group (P=0.03), DMEM Compared with the blank control group, the living cells of the control group (P<0.0001) and the glycerol group (P<0.0001) were significantly lower.
甘油组易造成解冻后组织水肿。Optisol能够在4℃下保存角膜内皮细胞长达2周，但更长期保存应用受限。DMEM因营养成分单一，难以长期维持天然角膜基质胶原超微结构和透光率特性。而本发明的营养胶囊组显著降低了储存成本，并提高了低温保存状态下细胞的存活率，可中长期保存角膜组织。The glycerol group tends to cause tissue edema after thawing. Optisol is capable of preserving corneal endothelial cells at 4°C for up to 2 weeks, but longer-term preservation is limited. Due to the single nutrient composition of DMEM, it is difficult to maintain the ultrastructure and light transmittance characteristics of natural corneal stroma collagen for a long time. However, the nutrient capsule group of the present invention significantly reduces the storage cost, improves the survival rate of cells in a low-temperature storage state, and can preserve corneal tissue in the medium and long term.
进一步对对比例的保存方案进行考查，并将对比例1-5用于中长期保存角膜基质透镜的结果与营养胶囊组(实施例1)进行比较，实验结果如下表1所示。Further examine the preservation plan of the comparative examples, and compare the results of comparative examples 1-5 for medium and long-term preservation of the corneal stroma lens with the nutrition capsule group (Example 1). The experimental results are shown in Table 1 below.
表1不同保存方法对角膜保存效果的影响Table 1 The effect of different preservation methods on the preservation effect of cornea
注：轻度水肿/空化泡：与新鲜组织相比出现＜30％水肿/空泡；Note: Mild edema/cavitation bleb: <30% edema/cavitation compared to fresh tissue;
中度水肿/空化泡：与新鲜组织相比出现30％-50％水肿/空泡；Moderate edema/cavitation bleb: 30%-50% edema/cavitation compared to fresh tissue;
重度水肿/空化泡：与新鲜组织相比出现＞50％水肿/空泡。Severe edema/cavitation: >50% edema/cavitation compared to fresh tissue.
从表1可以看出，采用对比例2-5中的保存方法用于保存角膜基质透镜，经过中长期保存后，角膜基质透镜的透明度、角膜基质细胞的活性均出现明显下降，而角膜基质透镜的纤维结构也发生明显破坏，其保存效果显著低于营养胶囊保存组。It can be seen from Table 1 that the preservation method in Comparative Examples 2-5 is used to preserve the corneal stroma lens. After medium and long-term preservation, the transparency of the corneal stroma lens and the activity of corneal stroma cells all appear to decline significantly, while the corneal stroma lens The fiber structure of the nutrient capsules was also significantly damaged, and its preservation effect was significantly lower than that of the nutrient capsule preservation group.
2、营养胶囊保存后角膜透镜组织结构及透光率检测2. Detection of corneal lens tissue structure and light transmittance after nutrition capsule storage
利用酶标仪对透镜的透光率进行检测。从图7中e分图可见，与对照组相比，7天后甘油组的平均透光率值显著降低(P＝0.013)，Optisol、DMEM和营养胶囊组保存的透镜透光率与新鲜透镜无显著差异。营养胶囊组存储14天后透镜透光率与对照组相比差异无统计学意义(P＝0.12)，其他3组透光率均低于对照组，差异有统计学意义(均P<0.05)。28天时，各组透光率均显著低于对照组(均P<0.05)。The light transmittance of the lens was detected by a microplate reader. As can be seen from the sub-graph e in Figure 7, compared with the control group, the average light transmittance value of the glycerin group decreased significantly after 7 days (P=0.013), and the light transmittance of the lenses preserved in the Optisol, DMEM and nutritional capsule groups had no difference with that of the fresh lenses. Significant differences. After storage for 14 days, there was no statistically significant difference in lens light transmittance between the nutritional capsule group and the control group (P=0.12), and the light transmittance of the other three groups was lower than that of the control group, and the difference was statistically significant (all P<0.05). At 28 days, the light transmittance of each group was significantly lower than that of the control group (all P<0.05).
此外，采用HE、MASSON、VG染色以及透射电镜来观察营养胶囊对透镜组织结构的保护作用。HE、Masson和VG染色结构显示，光镜下观察新鲜透镜角膜胶原纤维排列规则，边缘可见部分线性深染的角膜变性组织，组织深部未见明显损伤。保存7天后，营养胶囊组和Optisol组保存的透镜胶原纤维平行排列，整齐较规则，边界清晰。甘油组和DMEM组部分胶原纤维出现水肿，透镜的中间区可见少量的卵圆形的空化气泡。保存28天后，营养胶囊组和Optisol组透镜表面相对光滑，稍不规则，透镜的中间区可见少量的卵圆形的空化气泡，部分气泡存在相互连通。DMEM组和甘油组透镜边缘粗糙，胶原组织整体松散紊乱，纤维排列不平行，中心可见大的空化泡，部分空化泡相互融合。In addition, HE, MASSON, VG staining and transmission electron microscopy were used to observe the protective effect of nutritional capsules on lens tissue structure. HE, Masson, and VG staining structures showed that the collagen fibers of the fresh lens cornea were arranged regularly under light microscopy, and some linear deep-dyed corneal degeneration tissue could be seen at the edge, and no obvious damage was seen in the deep tissue. After being stored for 7 days, the lens collagen fibers preserved in the nutritional capsule group and the Optisol group were arranged in parallel, orderly and regular, with clear boundaries. In the glycerol group and the DMEM group, some collagen fibers appeared edema, and a small amount of oval cavitation bubbles could be seen in the middle area of the lens. After being stored for 28 days, the lens surfaces of the nutrition capsule group and the Optisol group were relatively smooth and slightly irregular, and a small amount of oval cavitation bubbles could be seen in the middle area of the lens, and some of the bubbles were interconnected. In the DMEM group and the glycerin group, the lens edge was rough, the collagen tissue was loose and disordered as a whole, the fibers were not arranged in parallel, large cavitation bubbles could be seen in the center, and some cavitation bubbles fused with each other.
采用透射电镜测定透镜胶原纤维密度。图7中d分图显示透镜的胶原原纤维直径在所有保存实验组和对照组间无显著差异。保存7天和14天后，所有实验组的平均角膜胶原原纤维数量与对照组相比无显著差异(P>0.05)。保存2天后，所有保存实验组的平均角膜胶原原纤维平均数量较对照组显著减少，差异有统计学意义。28天时，营养胶囊组平均纤维密度显著高于DMEM组(P＝0.016)和甘油组(P<0.001)，与Optisol组比较无显著性差异(图7中f分图)。与其它实验组相比，营养胶囊保存透镜组角膜胶原纤维排列更为紧密，空化气泡较少，透镜边缘清晰，有利于长期维持角膜透镜原有的胶原纤维结构和透明性。The density of lens collagen fibers was measured by transmission electron microscopy. Part d of Figure 7 shows that there is no significant difference in the diameter of the collagen fibrils of the lens between all preservation experimental groups and the control group. After storage for 7 days and 14 days, the average number of corneal collagen fibrils in all experimental groups had no significant difference compared with the control group (P>0.05). After 2 days of storage, the average number of corneal collagen fibrils in all preservation experimental groups was significantly reduced compared with that of the control group, and the difference was statistically significant. On day 28, the average fiber density of the nutrition capsule group was significantly higher than that of the DMEM group (P=0.016) and the glycerol group (P<0.001), and there was no significant difference compared with the Optisol group (part f in Figure 7). Compared with other experimental groups, the corneal collagen fibers in the lens group preserved in nutritional capsules were arranged more tightly, with less cavitation bubbles and clear lens edges, which was conducive to maintaining the original collagen fiber structure and transparency of the corneal lens for a long time.
上述结果均证实，营养胶囊保存后的角膜透镜组织具有良好的透光率及组织结构。The above results all confirmed that the corneal lens tissue after preservation in the nutritional capsule has good light transmittance and tissue structure.
3、营养胶囊保存后角膜基质透镜免疫原性检测3. Detection of immunogenicity of corneal stroma lens after preservation of nutritional capsules
经实验发现，各组透镜经保存后HLA-DR，HLA-ABC和CD45较少表达或不表达，说明角膜基质透镜经保存后免疫原性降低甚至消失。It was found through experiments that HLA-DR, HLA-ABC and CD45 were less expressed or not expressed in each group of lenses after preservation, indicating that the immunogenicity of corneal stromal lenses was reduced or even disappeared after preservation.
实验例4Experimental example 4
本实验例考查经营养胶囊保存的角膜基质透镜用于角膜透镜移植手术治疗角膜病变的效果。This experiment examines the effect of corneal stromal lens preserved in nutrient capsules on corneal lens transplantation for the treatment of corneal lesions.
(一)实验方法(1) Experimental method
采用营养胶囊包裹保存的透镜进行异体角膜组织移植治疗10例角膜病变患者(包括角膜营养不良、角膜变性、近视激光术后角膜扩张)，具体方法如下：Allogeneic corneal tissue transplantation was used to treat 10 patients with corneal lesions (including corneal dystrophy, corneal degeneration, and corneal ectasia after myopia laser surgery) with lenses wrapped in nutritional capsules. The specific methods are as follows:
透镜移植手术前，取出置于培养基中的营养胶囊，将透镜胶囊浸泡于3mL C溶液中以溶解胶囊壳层，并用3mLPBS洗涤3次，储存的供体角膜透镜从营养胶囊中释放，取出透镜后用PBS冲洗后，再用生理盐水冲洗后使用。Before the lens transplantation operation, take out the nutritional capsules placed in the medium, soak the lens capsules in 3mL C solution to dissolve the capsule shell, and wash with 3mL PBS for 3 times, the stored donor corneal lens is released from the nutritional capsules, and the lens is taken out After washing with PBS, rinse with saline before use.
术后随访时间分别为术后1天、1周、1月和3月，检查包括裂隙灯生物显微镜、未矫正远距离视力(UDVA)、主觉验光、矫正远距离视力(CDVA)、角膜地形图Pentacam眼前节断层扫描(Oculus)和眼前节AS-OCT。The postoperative follow-up time was 1 day, 1 week, 1 month and 3 months after operation, and the examinations included slit lamp biomicroscopy, uncorrected distance visual acuity (UDVA), subjective refraction, corrected distance visual acuity (CDVA), corneal topography Figure Pentacam anterior segment tomography (Oculus ) and anterior segment AS-OCT.
(二)实验结果(2) Experimental results
将在营养胶囊中保存28天的透镜取出后进行透镜异体移植用于治疗角膜疾病。首先对保存后的一部分透镜进行了微生物培养，确认保存后透镜未受到真菌及细菌污染后，进行了透镜移植手术。在本实验中，共纳入10例接收透镜异体移植的患者，10例患者中9眼行PTK-EP手术，1眼行LIKE手术。所有患者均完成了术后至少3个月的随访。手术中和术后随访患者均平安无事，无并发症或免疫排斥反应。The lenses stored in nutritional capsules for 28 days were taken out and used for lens allograft transplantation for the treatment of corneal diseases. Firstly, some of the preserved lenses were cultured for microorganisms, and after confirming that the preserved lenses were not contaminated by fungi and bacteria, lens transplantation was performed. In this experiment, a total of 10 patients who received lens allograft transplantation were included. Among the 10 patients, 9 eyes underwent PTK-EP surgery, and 1 eye underwent LIKE surgery. All patients completed at least 3 months of follow-up. During the operation and postoperative follow-up, all patients were safe and sound, without complications or immune rejection.
从图8显示，PTK-EP术后1天，角膜上皮开始生长，移植透镜贴附良好。术后1月，角膜上皮已完成重塑，透镜保持良好状态。术后随访期间，手术中所使用的保存透镜均保持透明。AS-OCT显示植入的透镜在整个随访期间显示出低反射率和可见的分界线。植入的透镜无移位、无皱纹，与相邻组织紧密粘附。随访期间，透镜周围组织密度增加，透镜与原本角膜组织间的分界线逐渐模糊。综上，这些结果证明经水凝胶营养胶囊保存后的透镜再利用进行异体移植具有良好的安全性及有效性。As shown in Figure 8, the corneal epithelium began to grow 1 day after PTK-EP, and the transplanted lens was well attached. One month after the operation, the corneal epithelium had been remodeled and the lens remained in good condition. During postoperative follow-up, the preservation lenses used during surgery remained clear. AS-OCT revealed that the implanted lenses showed low reflectivity and visible demarcation lines throughout the follow-up period. The implanted lens has no displacement, no wrinkle, and tight adhesion to adjacent tissues. During the follow-up period, the tissue density around the lens increased, and the boundary between the lens and the original corneal tissue gradually blurred. In summary, these results prove that the reuse of lenses preserved in hydrogel nutrient capsules for allograft transplantation has good safety and efficacy.
如图9所示，可以看出本发明构建的营养胶囊的结构与人体泪液结构组成具有高度相似性，在用于人体角膜组织如角膜基质透镜的保存时，具备维持人体泪液稳态的功能，能够起到很好的物理阻隔、营养供给和表面润滑等多重作用，可实现角膜基质透镜的中长期保存。As shown in Figure 9, it can be seen that the structure of the nutritional capsule constructed by the present invention is highly similar to the structure and composition of human tears, and when used for the preservation of human corneal tissues such as corneal stroma lenses, it has the function of maintaining the homeostasis of human tears. It can play multiple functions such as good physical barrier, nutrient supply and surface lubrication, and can realize medium and long-term preservation of corneal stromal lenses.
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