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

【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 lentivirusmediated 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 cellproliferation. 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.