钠泵抑制剂哇巴因对ECV304细胞连接相关分子表达的影响

Effect of Na-K-ATPase inhibitor ouabain on expression of cell junction-related molecules in ECV304 cells

目的:探讨钠泵抑制剂哇巴因对细胞连接相关分子表达的影响。方法:以不同浓度哇巴因作用ECV304细胞,用倒置相差显微镜和透射电镜观察细胞形态变化;通过四甲基偶氮唑盐(MTT)法检测细胞生长抑制作用;用Hoechst33342/PI双荧光染色、荧光显微镜分析细胞死亡特征;应用半定量逆转录聚合酶链反应(RT-PCR)检测细胞连接相关分子钠泵β1亚单位、内皮钙黏蛋白、P120连环蛋白1A和P120连环蛋白3AmRNA的表达。结果:哇巴因以浓度和作用时间依赖的方式抑制ECV304细胞生长;10μmol/L哇巴因作用24h,引起细胞坏死;0.1μmol/L哇巴因作用24～48h,细胞明显脱落,细胞间连接丧失,细胞出现染色质凝集、边移、凋亡小体形成等凋亡特征。RT-PCR结果显示钠泵β1亚单位、内皮钙黏蛋白和P120连环蛋白1A表达下降,P120连环蛋白3A表达上调。结论:哇巴因调节ECV304细胞连接相关分子表达从而影响其黏附和存活。

Abstract Objective： To study the effect of Na-K-ATPase inhibitor ouabain on the expression of cell junction-related molecules in ECV304 cells. Methods： ECV304 cells were treated with different concentration of ouabain. The morphologic changes were observed under a light microscope and a transmission electron microscope; The growth inhibition of cells was assayed by methyl-tetrazolium （MTT） assay; The feature of cell death was studied by Hoechst33342/PI staining and the mRNA expressions of Na-K-ATPase β1-subunit, VE-Cadherin, P120-catenin 1A and P120-catenin 3A were assayed by reverse transcription PCR （RT PCR）. Results： Ouabain inhibited the growth of ECV304 cells in a dose and time-dependent manner. 10 μmol/L ouabain treated for 24 hours could stimulate the necrosis of ECV304 cells; When treated with 0.1 μmol/L ouabain for 24-48 hours, the cells showed obviously deattachment, the loss of cell junction, nuclear chromatin condensation, margination and apoptotic bodies. The results of RT-PCR showed that the mRNA expressions of Na-K-ATPase 131-subunit, VE cadherin and P120 catenin 1A decreased, while the mRNA expression of P120-catenin 3A was significantly upregulated. Conclusion： Ouabain could affect cell adhesion and survival of ECV304 cells which was associated with the expression of cell junction-related molecules.