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

STIM1/ORAI1 requlates Ca^(2+) entry and NO generation mediated by human umbilical vein endothelial cells SOC and ROC

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Ca^(2+)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干扰质粒分别转染入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[Ca^(2+)]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干扰质粒同时转染入HUVECs,与Ca R激动剂精胺孵育后检测[Ca^(2+)]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,m RNA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[Ca^(2+)]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[Ca^(2+)]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Ca R激动剂的剌激下相互作用增强。由此可知,STIM1与ORAI1复合体共同调节Ca R经SOC和ROC激活介导的Ca^(2+)内流和NO生成。

To explore the function of STIM1/ORAI1 in store and receptor-operated Ca^（2＋）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 m RNA levels were determined by Western blotting and real-time PCR,respectively.The cells were incubated with four different treatment, intracellular Ca^（2＋）concentration（[Ca^（2＋）]i） was detected using the fluorescence Ca^（2＋）indicator Fura-2/AM,the production of NO was determined by DAF-FM of every group in HUVECs.HUVECs were transduced with sh RNA-STIM1 and sh RNA-ORAI1 at same time,after cultured with Ca R agonist, [Ca^（2＋）]iand the production of NO was determined.The interaction between ORAI1 and STIM1 were examined by Co-immunoprecipitation.Results：（1）Compared with control group,sh RNA targeted to the STIM1 or ORAI1 genes decreased their m RNA and protein levels,respectively（P0.05）;（2）In four different treatment under the action of factors,the [Ca^（2＋）]iand the net NO fluorescence intensity ratio values of transfection of STIM1 or ORAI1 sh RNA group were significantly reduced（P0.05）.（3）Compared with the control and single sh RNA group,the[Ca^（2＋）]iand the net NO fluorescence intensity ratio values of transfection of STIM1 and ORAI1 shRNA group were significantly reduced（P0.05）.（4）ORAI1 co-precipitates with STIM1,indicating internation of a molecular complex had enhanced by Ca R agonist.STIM1,ORAI1 are components of SOCE and ROCE channels in store and receptor-operated Ca^（2＋）entry and nitric oxide generation in human umbilical vein endothelial cells.