过表达Claudin-5增强体外培养的人视网膜微血管内皮细胞屏障功能  

Over-expression of Claudin-5 Enhanced the In Vitro Barrier Function of Human Retinal Vascular Endothelial Cell

作　　者：田蓉[1] 刘秋慧[1] 蔡萌[1] 李静[1] 王菁[1] 肖伟[1] 罗燕[1] 

机构地区：[1]中山大学中山眼科中心//眼科学国家重点实验室,广东广州510060

出　　处：《中山大学学报（医学科学版）》2014年第5期695-701,共7页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(30872819;81170864);教育部新世纪优秀人才支持计划(NCET-09-0809);中山大学青年教师培育项目(10ykpy27)

摘　　要：[目的]探讨过表达Claudin-5后对视网膜血管内皮细胞屏障功能的影响.[方法]体外原代培养人视网膜血管内皮细胞（hRVEC）.当接种至transwell膜的P2～P5代hRVEC近60％融合时进行慢病毒介导的转染实验.hRVEC分为对照组,慢病毒空载体组,慢病毒介导的Claudin-5高表达转染组.荧光活细胞动态显微镜观察慢病毒介导的转染效率.Western Blot检测各组细胞中Claudin-5表达水平.CCK8法检测病毒转染对细胞的毒性.待接种至transwell膜的hRVEC培养2周后建立稳定的单层人视网膜血管内皮细胞屏障模型后,电阻仪检测hRVEC屏障的跨内皮电阻（TER）,异硫氰酸荧光素右旋糖酐检测hRVEC屏障的通透性.[结果]慢病毒可以介导Claudin-5在hRVEC高表达,其转染率达60％,Western Blot检测证实Claudin-5蛋白表达显著增加.CCK8检测表明慢病毒转染不影响细胞的活性,对细胞毒性小;过表达Claudin-5后也不影响hRVEC的增殖;过表达Claudin-5后能降低体外单层人视网膜血管内皮细胞屏障模型的通透性,并提高其TER.[结论]过表达Claudin-5后可以显著提高体外人视网膜血管内皮细胞屏障的功能,这为视网膜血管性疾病的治疗提供新思路.[Objective] To investigate the effect of over-expression of Claudin-5 on the in vitro barrier function of human retinal vascular endothelial cell（hRVEC）.[Methods] hRVEC were isolated from human eyes and cultured.The second to fifth passage （P2-P5） hRVEC cultured on transwell membrane were conducted lentivirus-mediated transfection when the cells reached nearly 60％ confluent.hRVEC cultured on transwell membrane were devided into three groups：control group,empty lentivirus vector group and lentivirus-mediated Claudin-5 transfection group.Red fluorescein protein was used as a report gene,and the efficiency of lentivirus-mediated transfection was observed under fluorescence microscopy.The protein level of Claudin-5 in each group was detected by western blot.CCK8 assay was used to determine the cell viability after lentivirus transfection.Two weeks after seeding on the transwell membrane,hRVEC established a stable in vitro monolayer barrier model.Trans-endothelial resistance （TER） of hRVEC barrier was measured by resistance meter.Barrier permeability was measured by fluorescein isthiocyanate-dextran （FITC-Dextran）.[Results] Lentivirus could mediate the over-expression of Claudin-5 in hRVEC at about 60％ transfection efficiency.A significant increased protein level of Claudin-5 was found in the Claudin-5 transfection group.The cell viability in each group detected by CCK8 did not changed,indicating no cytotoxicity of lentivirus-mediated transfection,and no influence of over-expression of claudin-5 on cell proliferation.Over-expression of Claudin-5 significantly reduced the permeability of in vitro barrier model of hRVEC,and increased its TER.[Conclusion] Over-expression of Claudin-5 could significantly enhance the barrier functions of hRVEC.This study suggested over-expression of Claudin-5 might provide a new strategy for the treatment of retinal vascular diseases.

关 键 词：人视网膜血管内皮细胞 CLAUDIN-5 紧密连接 屏障功能 慢病毒转染 

分 类 号：R34[医药卫生—基础医学]