技术领域Technical Field
本发明涉及眼科药物技术领域，具体涉及一种立他司特在制备治疗视网膜动脉阻塞损伤的药物中的应用。The present invention relates to the technical field of ophthalmic drugs, and in particular to an application of lifalast in preparing a drug for treating retinal artery occlusion damage.
背景技术Background Art
视网膜动脉阻塞(Retinal Artery Occlusion，RAO)以无痛性、单侧视力的突然丧失为临床特征，由于血流的突然中断导致视网膜神经节细胞(Retinal Ganglion Cell,RGC)的大量死亡和强烈的炎症反应，尤其是小胶质细胞的活化，造成视觉功能丧失。当前，RAO的治疗措施主要包括溶栓治疗、高压氧治疗、降低眼内压和眼部按摩。在实际的临床工作中，大多数的患者已经错失溶栓治疗的时间窗，其他治疗措施虽然可以缓解一部分患者的症状，但是难以获得满意的视力改善。ROA致病因素复杂，临床缺少快速有效的治疗药物和方案。因此，研发价格低廉、治疗视网膜动脉损伤的药物具有十分重要的研究意义。Retinal artery occlusion (RAO) is characterized by painless, sudden unilateral vision loss. The sudden interruption of blood flow leads to massive death of retinal ganglion cells (RGC) and strong inflammatory response, especially activation of microglia, resulting in loss of visual function. Currently, the treatment measures for RAO mainly include thrombolytic therapy, hyperbaric oxygen therapy, lowering intraocular pressure and eye massage. In actual clinical work, most patients have missed the time window for thrombolytic therapy. Although other treatment measures can relieve the symptoms of some patients, it is difficult to achieve satisfactory vision improvement. The pathogenic factors of ROA are complex, and there is a lack of rapid and effective treatment drugs and programs in clinical practice. Therefore, it is of great research significance to develop low-cost drugs for the treatment of retinal artery damage.
立他司特(Lifitegrast)是一种新型小分子整合素拮抗剂，可以人工合成的化合物，分子式为C29H24Cl2N2O7S，CAS号为1025967-78-5，结构式如下：Lifitegrast is a new type of small molecule integrin antagonist, a synthetic compound with a molecular formula of C29 H24 Cl2 N2 O7 S, CAS No. 1025967-78-5, and the structural formula is as follows:
有研究已经证实，立他司特是一种新的细胞间黏附因子的抑制剂，可以通过阻断细胞间黏附分子-1和整合素蛋白淋巴细胞功能相关抗原-1之间的结合起效。2016年7月，美国食品药品管理局(FDA)正式批准了5％立他司特滴眼剂(商品名XiidraTM)的申请，是FDA批准的第一个可以改善和治疗干眼症症状的新药。该药的临床试验主要包括针对干眼症患者的1个为期12周的II期临床试验和3个为期12周的III期临床试验，研究结果充分证明了该药的有效性和安全性。Studies have confirmed that lifastat is a new inhibitor of intercellular adhesion factors, which can work by blocking the binding between intercellular adhesion molecule-1 and integrin protein lymphocyte function-associated antigen-1. In July 2016, the U.S. Food and Drug Administration (FDA) officially approved the application of 5% lifastat eye drops (trade name XiidraTM), which is the first new drug approved by the FDA to improve and treat dry eye symptoms. The clinical trials of this drug mainly include a 12-week Phase II clinical trial and three 12-week Phase III clinical trials for patients with dry eye. The results of the study fully demonstrated the effectiveness and safety of the drug.
视网膜动脉阻塞损伤属于眼底病范畴，目前尚无有效的治疗药物，立他司特在该疾病中尚无研究报道，因此有必要研发治疗视网膜动脉阻塞损伤的新型治疗药物。Retinal artery occlusion damage belongs to the category of fundus diseases. There is currently no effective treatment for this disease. There are no research reports on levofloxacin in this disease. Therefore, it is necessary to develop new therapeutic drugs for the treatment of retinal artery occlusion damage.
本发明拟研发立他司特在视网膜动脉阻塞损伤中的新用途，为临床研究提供治疗手段和思路。The present invention intends to develop a new use of lifalast in retinal artery occlusion injury, and provide treatment methods and ideas for clinical research.
发明内容Summary of the invention
本发明的目的是提供一种立他司特的新用途，以解决视网膜动脉阻塞的治疗手段匮乏难题。通过实验发现立他司特能有效减轻小鼠视网膜动脉阻塞损伤和视网膜神经节细胞的凋亡。同时，立他司特还具有抑制视网膜的炎症浸润和小胶质细胞的活化的作用。同时，立他司特还具有减轻视网膜神经节细胞损伤的作用，可以用于视网膜动脉阻塞的神经炎症损伤等相关疾病，具有广泛的应用前景。The purpose of the present invention is to provide a new use of lifalast to solve the problem of lack of treatment methods for retinal artery occlusion. Through experiments, it was found that lifalast can effectively reduce the damage of retinal artery occlusion and apoptosis of retinal ganglion cells in mice. At the same time, lifalast also has the effect of inhibiting retinal inflammatory infiltration and activation of microglia. At the same time, lifalast also has the effect of reducing retinal ganglion cell damage, can be used for related diseases such as neuroinflammatory damage caused by retinal artery occlusion, and has broad application prospects.
本发明的目的通过下述技术方案：The purpose of the present invention is achieved through the following technical solutions:
一方面，本发明的目的是提供一种立他司特在制备治疗视网膜动脉阻塞损伤的药物中的应用。On the one hand, the purpose of the present invention is to provide a use of lifalast in the preparation of a medicament for treating retinal artery occlusion damage.
第二方面，本发明的目的是提供一种含立他司特的组合物在制备治疗视网膜动脉阻塞损伤的药物中的应用。In a second aspect, the present invention aims to provide a use of a composition containing lifalast in the preparation of a medicament for treating retinal artery occlusion damage.
第三方面，本发明的目的是提供一种立他司特在制备抑制Inos、Tnf-α、Il-1β、IL-6中的至少一种的基因表达的药物中的应用。In a third aspect, the present invention aims to provide a use of lifalast in the preparation of a drug for inhibiting the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6.
第四方面，本发明的目的是提供一种含立他司特的组合物在制备抑制Inos、Tnf-α、Il-1β、IL-6中的至少一种的基因表达的药物中的应用。In a fourth aspect, the present invention aims to provide a composition containing lifalast for use in the preparation of a drug for inhibiting the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6.
第五方面，立他司特在制备减轻视网膜神经节细胞细胞凋亡的药物中的应用。Fifthly, use of lifalast in the preparation of a drug for alleviating apoptosis of retinal ganglion cells.
所述药物的给药方式为：玻腔注射给药。The administration method of the drug is: intravitreal injection.
给药剂量为0.1-1μg。The dosage is 0.1-1 μg.
所述药物的作用对象为小胶质细胞系BV2或视网膜前体细胞系R28。The drug acts on the microglial cell line BV2 or the retinal progenitor cell line R28.
第六方面，本发明的目的是提供一种治疗视网膜动脉阻塞损伤的药物，包含立他司特。In a sixth aspect, the present invention aims to provide a drug for treating retinal artery occlusion damage, comprising lifalast.
第七方面，本发明的目的是提供一种抑制Inos、Tnf-α、Il-1β、IL-6中的至少一种的基因表达的药物，包含立他司特。In a seventh aspect, an object of the present invention is to provide a drug for inhibiting the gene expression of at least one of Inos, Tnf-α, Il-1β, and IL-6, comprising lifalast.
还包含药学上可接受的盐与药学上可接受的载体组成临床上可接受的药物或制剂。It also comprises pharmaceutically acceptable salts and pharmaceutically acceptable carriers to form clinically acceptable drugs or preparations.
所述载体为药学领域的常规稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂、香剂、甜味剂中的至少一种。The carrier is at least one of conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, flavoring agents, and sweeteners in the pharmaceutical field.
本发明具有以下优点和有益效果：The present invention has the following advantages and beneficial effects:
本发明发现立他司特的新用途，以解决视网膜动脉阻塞的治疗手段匮乏难题。立他司特能有效减轻视网膜动脉阻塞损伤和视网膜神经节细胞的凋亡。同时，立他司特还具有抑制视网膜的炎症浸润和小胶质细胞的活化的作用。The present invention discovers a new use of Litahist to solve the problem of lack of treatment methods for retinal artery occlusion. Litahist can effectively reduce retinal artery occlusion damage and apoptosis of retinal ganglion cells. At the same time, Litahist also has the effect of inhibiting retinal inflammatory infiltration and microglial activation.
立他司特还具有减轻视网膜神经节细胞损伤的作用，可以用于视网膜动脉阻塞的神经炎症损伤等相关疾病，具有广泛的应用前景。Lifalast also has the effect of reducing retinal ganglion cell damage and can be used for related diseases such as neuroinflammatory damage caused by retinal artery occlusion, and has broad application prospects.
本发明中立他司特具有抑制小胶质细胞活化的作用，该作用的对象是小胶质细胞系BV2。The nilotamidate of the present invention has an effect of inhibiting microglial activation, and the target of the effect is the microglial cell line BV2.
本发明中立他司特具有缓解视网膜神经节细胞损伤的功能作用的对象是视网膜前体细胞系R28。The object of the functional effect of ritamilast in the present invention on alleviating retinal ganglion cell damage is the retinal progenitor cell line R28.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为实施例2的小鼠视网膜电图(ERG)检测情况；Fig. 1 is the detection situation of the mouse electroretinogram (ERG) of Example 2;
图2为实施例2的小鼠视网膜HE染色病理结果图片；FIG2 is a picture of the pathological results of HE staining of the mouse retina in Example 2;
图3为实施例2的小鼠视网膜铺片免疫荧光染色的RGC结果图片；FIG3 is a picture of the RGC results of immunofluorescence staining of mouse retinal flat mounts in Example 2;
图4为实施例3的不同浓度的立他司特对BV2细胞活性的影响；FIG4 shows the effects of different concentrations of lifalast on the activity of BV2 cells in Example 3;
图5为实施例3的立他司特对BV2细胞iNOS表达的影响；FIG5 shows the effect of Litamilast in Example 3 on the expression of iNOS in BV2 cells;
图6为实施例3的立他司特对BV2细胞TNF-α表达的影响；FIG6 shows the effect of Litamilast in Example 3 on TNF-α expression in BV2 cells;
图7为实施例3的立他司特对BV2细胞IL-1β表达的影响；FIG7 shows the effect of Litamilast of Example 3 on IL-1β expression in BV2 cells;
图8为实施例3的立他司特对BV2细胞IL-6表达的影响；FIG8 shows the effect of Litamilast in Example 3 on IL-6 expression in BV2 cells;
图9为实施例4的立他司特对R28细胞氧糖剥夺损伤的的影响。FIG. 9 shows the effect of lifalast in Example 4 on oxygen-glucose deprivation injury in R28 cells.
具体实施方式DETAILED DESCRIPTION
为更好的理解本发明，下面的实施例是对本发明的进一步说明，但本发明的内容不仅仅局限于下面的实施例。For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。Now, various exemplary embodiments of the present invention are described in detail, which should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by conventional technicians in the field of the present invention. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which is obvious to those skilled in the art. Other embodiments obtained from the description of the present invention are obvious to technicians. The present invention description and examples are exemplary only. Regarding the "comprising", "including", "having", "containing" and the like used herein, they are all open terms, that is, they mean including but not limited to.
本发明提供了一种视网膜缺血再灌注损伤的小鼠模型，进一步地，该小鼠模型是通过手术方式将小鼠的翼腭动脉进行阻塞一段时间后进行血流再通以模拟疾病的病理过程。进一步地，小鼠翼腭动脉阻塞的时间为2小时，血液再灌注时间为7天。进一步地，阻塞翼腭动脉是采用线栓的方法实现的。本发明还研发一种立他司特治疗小鼠视网膜动脉阻塞损伤的方法，进一步地，该方法的给药方式为玻腔注射，给药浓度为1ug/ul,体积为1ul，给药时间为每两天给药一次。本发明还提供一种立他司特的抑制小胶质细胞活化的作用，进一步地，该作用的对象是小鼠小胶质细胞系BV2。本发明还提供一种立他司特的缓解视网膜神经节细胞损伤的功能，该作用的对象是大鼠视网膜前体细胞系R28。The present invention provides a mouse model of retinal ischemia-reperfusion injury. Further, the mouse model is a mouse model in which the pterygopalatine artery of the mouse is blocked for a period of time by surgery and then blood flow is restored to simulate the pathological process of the disease. Further, the pterygopalatine artery of the mouse is blocked for 2 hours and the blood reperfusion time is 7 days. Further, the occlusion of the pterygopalatine artery is achieved by a thread plug method. The present invention also develops a method for treating retinal artery occlusion injury in mice with lifastat. Further, the method is administered by intravitreal injection, the administration concentration is 1ug/ul, the volume is 1ul, and the administration time is once every two days. The present invention also provides a function of lifastat in inhibiting microglia activation, and further, the object of the action is the mouse microglia cell line BV2. The present invention also provides a function of lifastat in alleviating retinal ganglion cell damage, and the object of the action is the rat retinal progenitor cell line R28.
实施例1：小鼠视网膜动脉阻塞损伤动物模型的建立：Example 1: Establishment of an animal model of retinal artery occlusion injury in mice:
准备8周龄的C57BL/6雄性小鼠，其体重严格控制在20-25g。然后准备气体麻醉装置。通过橡胶管用1.5-2％异氟烷的一氧化二氮/氧气混合物麻醉小鼠。术中体温维持在37±0.5℃。手术器械用75％酒精消毒。将小鼠仰卧在加热毯上并暴露其颈部。然后除去小鼠颈毛，消毒皮肤并沿颈部中线剪开皮肤。用两个镊子拉开颈腺。直截了当地分离左侧颈总动脉、颈内动脉和颈外动脉，但不要挤压紧贴的神经和静脉。然后用血管夹夹住颈总动脉和颈内动脉。用8-0丝线缝合在颈外动脉远端打结，近端打活结。用眼科剪刀在颈外动脉的两个缝合结之间做一个小切口，然后将特殊的线栓塞沿着切口插入颈外动脉和颈总动脉。接下来，取下颈内动脉夹，在颈外动脉开口处剪断颈外动脉，将线栓拉回颈总动脉分叉处，反转插入颈内动脉，翼腭动脉和眼动脉。将线栓插入翼腭动脉末端，距离分叉处约6毫米。线栓的尾部几乎在分叉处，堵塞了眼动脉。然后系紧活结并缝合皮肤。小鼠在动脉栓塞期间可以自由走动。栓塞120分钟后，小心地将线栓从翼腭动脉取出，无大出血。移除颈总动脉夹并恢复动脉再灌注。然后缝合皮肤伤口并在再灌注期间常规喂养小鼠。Prepare 8-week-old C57BL/6 male mice, whose weight is strictly controlled at 20-25g. Then prepare a gas anesthesia device. Anesthetize the mice with a nitrous oxide/oxygen mixture of 1.5-2% isoflurane through a rubber tube. The body temperature is maintained at 37±0.5℃ during the operation. The surgical instruments are disinfected with 75% alcohol. The mice are placed supine on a heating blanket and their necks are exposed. Then remove the hair on the neck of the mice, disinfect the skin and cut the skin along the midline of the neck. Pull open the neck gland with two forceps. Straightly separate the left common carotid artery, internal carotid artery and external carotid artery, but do not squeeze the closely attached nerves and veins. Then clamp the common carotid artery and internal carotid artery with a vascular clamp. Tie a knot at the distal end of the external carotid artery with 8-0 silk suture and a slipknot at the proximal end. Make a small incision between the two suture knots of the external carotid artery with ophthalmic scissors, and then insert a special line embolization into the external carotid artery and common carotid artery along the incision. Next, the internal carotid artery clamp was removed, the external carotid artery was cut at the opening of the external carotid artery, the suture was pulled back to the bifurcation of the common carotid artery, and reversed and inserted into the internal carotid artery, pterygopalatine artery, and ophthalmic artery. The suture was inserted into the end of the pterygopalatine artery, about 6 mm from the bifurcation. The tail of the suture was almost at the bifurcation, blocking the ophthalmic artery. The slipknot was then tied and the skin was sutured. The mice were free to move around during the arterial embolization. After 120 minutes of embolization, the suture was carefully removed from the pterygopalatine artery without major bleeding. The common carotid artery clamp was removed and arterial reperfusion was resumed. The skin wound was then sutured and the mice were fed routinely during reperfusion.
实施例2：立他司特对小鼠视网膜动脉阻塞损伤体内作用：Example 2: In vivo effects of lifalast on retinal artery occlusion injury in mice:
小鼠的玻璃体腔注射立他司特24小时后，进行小鼠视网膜动脉阻塞造模，7天后将小鼠暗适应12小时，进行小鼠视网膜电位测定；小心摘取小鼠眼球，置于专用眼球固定液2小时以上，进行石蜡包埋、切片和HE染色；同时，另一半小鼠在手术显微镜下小心分离视网膜，确保视网膜的完整和细胞活性，进行固定和Brn3a的免疫荧光染色，拍照分析。图1为实施例2的小鼠视网膜电图(ERG)检测情况，从图中可以看出：立他司特处理的小鼠的ERG中波幅更高、视网膜厚度较对照组增加，视网膜神经节密度增加，表明立他司特可以有效缓解视网膜动脉阻塞中视网膜神经节细胞的损伤，有助于视功能的恢复。24 hours after the intravitreal injection of lisdenafil, the mouse retinal artery occlusion model was established, and 7 days later, the mouse was dark-adapted for 12 hours, and the mouse retinal potential was measured; the mouse eyeball was carefully removed, placed in a special eyeball fixative for more than 2 hours, and paraffin-embedded, sliced and HE-stained; at the same time, the other half of the mice were carefully separated from the retina under a surgical microscope to ensure the integrity and cell activity of the retina, fixed and immunofluorescently stained for Brn3a, and photographed for analysis. Figure 1 is the detection of the mouse electroretinogram (ERG) of Example 2. It can be seen from the figure that the ERG of the mice treated with lisdenafil has a higher amplitude, the retinal thickness is increased compared with the control group, and the retinal ganglion density is increased, indicating that lisdenafil can effectively alleviate the damage of retinal ganglion cells in retinal artery occlusion and contribute to the recovery of visual function.
图2为实施例2的小鼠视网膜HE染色病理结果图片，从图中可以看出；立他司特组的小鼠视网厚度增加，损伤减轻。FIG. 2 is a picture of the pathological results of HE staining of the mouse retina in Example 2. It can be seen from the figure that the retinal thickness of the mice in the ritalidomide group increased and the damage was alleviated.
图3为实施例2的小鼠视网膜铺片免疫荧光染色的RGC结果图片，从图中可以看出；立他司特处理后视网膜中的神经节细胞数量增加。FIG3 is a picture of the RGC results of immunofluorescence staining of the mouse retina in Example 2. It can be seen from the figure that the number of ganglion cells in the retina increases after rifalast treatment.
实施例3：立他司特对小胶质细胞活化代谢的影响：Example 3: Effect of Lifalast on Microglial Activation and Metabolism:
将小鼠的小胶质细胞系BV2进行体外培养，在细胞生长的对数期进行铺板分组，分为对照组、细菌脂多糖(LPS)刺激组和LPS+立他司特组，LPS浓度为200ng/ml，立他司特浓度为100ng/ml。刺激24小时后，收集细胞，提取RNA，进行RNA的浓度和质量测定，行Real-TimePCR检测Inos、Tnf-α、Il-1β、IL-6的mRNA表达情况，分析相对表达水平变化。实验结果表明，立他司特可以有效抑制小胶质细胞的代谢和活化。The mouse microglial cell line BV2 was cultured in vitro, and the cells were plated and grouped in the logarithmic phase of cell growth, divided into a control group, a bacterial lipopolysaccharide (LPS) stimulation group, and an LPS+lifastat group. The LPS concentration was 200 ng/ml, and the lifastat concentration was 100 ng/ml. After 24 hours of stimulation, the cells were collected, RNA was extracted, and the concentration and quality of RNA were determined. Real-Time PCR was performed to detect the mRNA expression of Inos, Tnf-α, Il-1β, and IL-6, and the relative expression level changes were analyzed. The experimental results showed that lifastat can effectively inhibit the metabolism and activation of microglia.
图4为实施例3的不同浓度的立他司特对BV2细胞活性的影响，从图中可以看出；立他司特的浓度从0到100ng/ml都不影响BV2细胞的活性。FIG. 4 shows the effects of different concentrations of lifalast on the activity of BV2 cells in Example 3. It can be seen from the figure that the concentration of lifalast ranging from 0 to 100 ng/ml does not affect the activity of BV2 cells.
图5为实施例3的立他司特对BV2细胞iNOS表达的影响，从图中可以看出；立他司特可以抑制iNOS的基因表达水平，减少小胶质细胞活化。FIG5 shows the effect of lifalast in Example 3 on the expression of iNOS in BV2 cells. It can be seen from the figure that lifalast can inhibit the gene expression level of iNOS and reduce the activation of microglia.
图6为实施例3的立他司特对BV2细胞TNF-α表达的影响，从图中可以看出；立他司特可以抑制TNF-α的基因表达水平，减少小胶质细胞活化。FIG6 shows the effect of Litamilast in Example 3 on the expression of TNF-α in BV2 cells. It can be seen from the figure that Litamilast can inhibit the gene expression level of TNF-α and reduce the activation of microglia.
图7为实施例3的立他司特对BV2细胞IL-1β表达的影响，从图中可以看出；立他司特可以抑制IL-1β的基因表达水平，减少小胶质细胞活化。FIG7 shows the effect of Litamilast in Example 3 on the expression of IL-1β in BV2 cells. It can be seen from the figure that Litamilast can inhibit the gene expression level of IL-1β and reduce the activation of microglia.
图8为实施例3的立他司特对BV2细胞IL-6表达的影响，从图中可以看出；立他司特可以抑制IL-6的基因表达水平，减少小胶质细胞活化。FIG8 shows the effect of Litahist in Example 3 on the expression of IL-6 in BV2 cells. It can be seen from the figure that Litahist can inhibit the gene expression level of IL-6 and reduce the activation of microglia.
实施例4：立他司特对视网膜神经节细胞氧糖剥夺损伤的影响：Example 4: Effects of Lifalast on Oxygen-Glucose Deprivation Injury of Retinal Ganglion Cells:
将大鼠的视网膜神经节前体细胞系R28进行体外培养，在细胞生长的对数期进行铺板分组，进行氧糖剥夺处理，氧糖剥夺6小鼠，复氧24小时，分为溶剂对照组和立他司特组，立他司特浓度为100ng/ml。刺激时间为24小时。收集细胞，将细胞消化为单细胞悬液，用PBS重悬后，进行Annexin V和PI染色，进行流式细胞术分析细胞凋亡情况，图9为实施例4的立他司特对R28细胞氧糖剥夺损伤的的影响，发现立他司特处理后，凋亡细胞的比例显著下降。实验结果表明，立他司特可以有减轻视网膜神经节细胞的损伤。The rat retinal ganglion precursor cell line R28 was cultured in vitro, plated and grouped in the logarithmic phase of cell growth, and subjected to oxygen-glucose deprivation treatment. The mice were deprived of oxygen and glucose for 6 hours, and reoxygenated for 24 hours. They were divided into a solvent control group and a lifastat group, and the lifastat concentration was 100 ng/ml. The stimulation time was 24 hours. The cells were collected, digested into a single cell suspension, resuspended in PBS, stained with Annexin V and PI, and analyzed by flow cytometry for cell apoptosis. Figure 9 shows the effect of lifastat in Example 4 on oxygen-glucose deprivation damage of R28 cells. It was found that after lifastat treatment, the proportion of apoptotic cells decreased significantly. The experimental results show that lifastat can reduce the damage of retinal ganglion cells.
以上所述是本发明的优选实施方式而已，当然不能以此来限定本发明之权利范围，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和变动，这些改进和变动也视为本发明的保护范围。The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.
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| CN202411082094.1ACN118903130B (en) | 2024-08-08 | 2024-08-08 | Application of ritalst in preparation of medicine for treating retinal artery occlusion injury |
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| US20090298869A1 (en)* | 2008-04-15 | 2009-12-03 | John Burnier | Crystalline pharmaceutical and methods of preparation and use thereof |
| CN110381920A (en)* | 2017-03-09 | 2019-10-25 | 雅利斯塔制药公司 | Peptide for scheroma |
| US20200009130A1 (en)* | 2016-12-16 | 2020-01-09 | Sarcode Bioscience Inc. | Ocular distribution and pharmacokinetics of lifitegrast formulations |
| CN113795273A (en)* | 2019-04-22 | 2021-12-14 | 莎思坦控股有限责任公司 | Collagen peptide-based pharmaceutical composition and device, production method and use thereof |
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| US20090298869A1 (en)* | 2008-04-15 | 2009-12-03 | John Burnier | Crystalline pharmaceutical and methods of preparation and use thereof |
| US20200009130A1 (en)* | 2016-12-16 | 2020-01-09 | Sarcode Bioscience Inc. | Ocular distribution and pharmacokinetics of lifitegrast formulations |
| CN110381920A (en)* | 2017-03-09 | 2019-10-25 | 雅利斯塔制药公司 | Peptide for scheroma |
| CN113795273A (en)* | 2019-04-22 | 2021-12-14 | 莎思坦控股有限责任公司 | Collagen peptide-based pharmaceutical composition and device, production method and use thereof |
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| GUSTAVO ORTIZ等: "Topical Leukocyte Function-Associated Antigen-1 (LFA-1) Antagonist Treatment (Lifitegrast) Suggest that Immune Synapsis and T cell Adhesion in Limbal Vessels is affected during DED", 《INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE》, vol. 60, no. 9, 31 July 2019 (2019-07-31)* |
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