STIM1/ORAI1复合体参与调节人脐静脉内皮细胞SOC和ROC介导的钙内流和NO生成

摘要

To explore the function of STIM1/ORAI1 in store and receptor-operated Ca2+ entry and nitric oxide generation by SOC and ROC in human umbilical vein endothelial cells.HUVECs were collected and cultured to the second～third passage.We silenced the expression of their genes in HUVECs by transfection constructed STIM1 or ORAI1 RNA interference plasmids.The interference efficiency of their protein and mRNA levels were determined by Western blotting and real-time PCR,respectively.The cells were incubated with four different treatment,intracellular Ca2+ concentration ~Ca2~ was detected using the fluorescence Ca2+ indicator Fura-2/AM,the production of NO was determined by DAF-FM of every group in HUVECs.HUVECs were transduced with shRNA-STIM1 and shRNA-ORAI1 at same time,after cultured with CaR agonist,[Ca2+]i and the production of NO was determined.The interaction between ORAI1 and STIM1 were examined by Co-immunoprecipitation.Results:(1) Compared with control group,shRNA targeted to the STIM1 or ORAI1 genes decreased their mRNA and protein levels,respectively (P＜ 0.05);(2)In four different treatment under the action of factors,the [Ca2+]i and the net NO fluorescence intensity ratio values of transfection of STIM1 or ORAI1 shRNA group were significantly reduced (P＜0.05).(3)Compared with the control and single shRNA group,the[Ca2+]i and the net NO fluorescence intensity ratio values of transfection of STIM1 and ORAI1 shRNA group were significantly reduced (P＜ 0.05).(4)ORAI1 co-precipitates with STIM1,indicating internation of a molecular complex had enhanced by CaR agonist.STIM1,ORAI1 are components of SOCE and ROCE channels in store and receptor-operated Ca2+ entry and nitric oxide generation in human umbilical vein endothelial cells.%为研究基质交联分子1(STIM1)/钙释放激活钙通道调节分子1(ORAI1)复合体在人脐静脉内皮细胞(HUVECs)钙池操-纵性钙通道(SOC)和受体操纵性钙通道(ROC)介导Ca2+内流和NO生成中的作用.采取2-3代HUVECs随机分组,将构建的STIM1和ORAI1干扰质粒分别转染人HUVECs.用激光共聚焦显微镜观察细胞转染效果,Real-time PCR和Western blotting检测STIM1、ORAI1 mRNA和蛋白的表达.细胞随机分组:特异性质粒转染组即实验组,未转染组即空白对照组(Control组)及空质粒组(Scrambled组),将上述3组细胞分别与四种不同处理因素刺激后用荧光探针Fura-2/AM检测[Ca2+]i变化,NO荧光探针DAF-FM负载方法同步检测NO生成的变化.随后将构建的STIM1和ORAI1干扰质粒同时转染人HUVECs,与CaR激动剂精胺孵育后检测[Ca2+]i和NO,用免疫共沉淀法检测STIM1和ORAI1的相互作用.结果显示,(1)与对照组相比,STIM1及ORAI1组,mRNA和蛋白表达均明显降低(P＜0.05);(2)在4种不同处理因素作用下,STIM1及ORAI1转染组中[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P＜0.05);(3)与对照组及单转染STIM1及ORAI1组比,共转染组[Ca2+]i△ratio值和NO净荧光强度值均明显降低(P＜0.05);(4) STIM1与ORAI1相互作用形成复合体,且在CaR激动剂的刺激下相互作用增强.由此可知,STIM1与ORAI1复合体共同调节CaR经SOC和ROC激活介导的Ca2+内流和NO生成.