小干扰RNA沉默细胞外钙敏感受体抑制人脐静脉内皮细胞钙内流和NO生成

Extracellular Ca^(2+) influx and NO generation are inhibited by small interference RNA targeting extracellular Ca^(2+)-sensing receptor in human umbilical vein endothelial cells

为了探讨细胞外钙敏感受体(extracellular Ca^(2+)-sensing receptor, CaR)在人脐静脉内皮细胞(human umbilical veinendothelial cells, HUVEC)介导钙内流和NO生成中的作用,本实验针对GenBank中人CaR的cDNA序列设计合成小干扰RNA(small interference RNA, siRNA),并将其转染入HUVEC中,采用激光共聚焦显微镜观察细胞转染效果,Western blot检测CaR蛋白表达及转染后的干扰效率,Fura-2/AM负载检测细胞内Ca^(2+)浓度(intracellular Ca^(2+)concentration, [Ca^(2+)]i)变化,NO荧光探针DAF-FM DA负载检测细胞内NO的生成及内皮型一氧化氮合酶(endothelial nitric oxide synthase, eNOS)活性。结果显示,实验成功将CaR siRNA转入HUVEC,Western blot 检测结果显示转染48 h后,CaR蛋白表达降低(P < 0.05);与未转染对照组和阴性对照组相比,在精胺 + Ca^(2+)刺激下,CaR siRNA转染组的[Ca^(2+)]i、eNOS活性和NO含量均显著降低(P < 0.05)。上述结果表明,CaR在精胺介导的HUVEC Ca^(2+)内流和NO生成中发挥重要作用。

To investigate the effect of Ca2＋-sensing receptor（CaR） on Spermine-induced extracellular Ca2＋ influx and NO generation in human umbilical vein endothelial cells（HUVEC）,the small interference RNA（siRNA） specifically targeting CaR gene was designed,synthesized and transfected into HUVEC according to the cDNA sequence of human CaR gene in GenBank.The transfection efficiency and the interference efficiency of CaR protein were determined by laser scanning confocal microscopy and Western blot,respectively.Intracellular Ca2＋ concentration（[Ca2＋ ] i） was measured by Fura-2/AM loading.The production of NO and the activity of endothelial nitric oxide synthase（eNOS） were determined by the DAF-FM diacetate（DAF-FM DA）.Western blot results demonstrated that siRNA targeting the CaR specifically decreased the expression of CaR protein in CaR siRNA group 48 h after transfection（P 〈 0.05）.At the same time,the Spermine-induced [Ca2＋ ] i,eNOS activity and NO generation were also significantly reduced（P 〈 0.05） in CaR siRNA group compared with those in the untransfected or negative siRNA transfected group.In conclusion,the present study suggests that the CaR plays an important role in the Spermine-evoked process of extracellular Ca2＋ influx and NO generation in HUVEC.