LPS对内皮细胞纤维状肌动蛋白和钙依赖性粘附素的细胞定位和结构的影响

目的:通过观察人脐静脉内皮细胞的纤维状肌动蛋白(F-actin)和内皮细胞钙依赖性粘附素(VE-cadherin)的细胞定位和结构变化,从形态学的角度阐述LPS对内皮细胞的作用。方法:培养人脐静脉内皮细胞,选用VE-cadherin的特异性抗体,采用免疫荧光技术观察不同浓度LPS刺激下VE-cadherin的胞膜定位和结构的变化;选用F-actin的特异性结合物鬼笔环肽进行荧光染色,观察不同浓度LPS刺激下F-actin细胞定位和结构的变化。结果:正常状态下VE-cadherin位于内皮细胞间连接处,染色轮廓清晰光滑,细胞间无明显缝隙。较高浓度LPS刺激后可看到细胞间连接处染色减少,呈锯齿状,部分视野细胞回缩,有明显的细胞间隙形成。正常组F-actin主要分布在细胞的周边以及胞核周围,分布均匀、排列整齐。较高浓度LPS刺激后,内皮细胞周边的F-actin基本消失,出现明显的应力纤维(变粗、变短、紊乱,排列呈明显的极向分布),可见丝状和板状伪足。结论:高浓度的LPS(大于300μg/L)作用于内皮细胞,可影响F-actin和VE-cadherin的结构重组和细胞内分布。

Effects of LPS on the organization and localization of VE-cadherin and F-actin in cultured endothelial cells

AIM:This experiment is to investigate the effects of LPS on the organization and localization of VE-cadherin and F-actin in cultured human umbilical endothelial cells.METHODS:The human umbilical vein endothelial cell lines ECV-304 were incubated with LPS at different concentrations for 30 min. VE-cadherin was detected by immunofluorescence with primary mAb of VE-cadherin and FITC-conjugated secondary antibody. F-actin was detected with fluorescence staining with rodamine-phalloidin.RESULTS:At high concentration, LPS could induce reorganization of VE-cadherin with the formation of serrata cellular border and enlargement of intercellular gaps, which were apparently different from that in normal conditions with the high fluorescence intensity at cell-cell junction. F-actin depolymerization could also be induced by LPS at high concentration with the formation of stress fiber and filopodia.CONCLUSION:LPS(300 μg/L) could induce reorganization of VE-cadherin and F-actin in human umbilical vein endothelial cells.