人脐静脉内皮细胞中C-型瞬时型感受器电位通道3RNA干扰质粒构建及筛选

The Construction and Screening of RNA Interference Plasmid of C-Transient Receptor Potential Canonical-3 in Human Umbilical Vein Endothelial Cells

为了探讨C-型瞬时型感受器电位通道3(TRPC3)在人脐静脉内皮细胞(HUVEC)中的作用,本实验针对GenBank中人TRPC3的cDNA序列设计合成短发卡RNA(shRNA),将其转染到HUVEC中,在激光共聚焦显微镜下观察细胞的转染效率,实时定量PCR和免疫印记检测TRPC3mRNA和蛋白表达及转染后的干扰效率。结果显示:(1)激光共聚焦显微镜下观察到绿色荧光,说明已成功将shTRPC3转入到HUVEC中;(2)转染48h后用G418进行稳定筛选后,TRPC3mRNA和蛋白表达均降低(P<0.05),干扰效率分别为71%和74%。由此可知,在HUVEC中成功构建了TRPC3干扰质粒。

To investigate the effect of C-Transient Receptor Potential Canonical-3 （TRPC3） in human umbilical vein endothelial cells （HUVEC） ,this experiment,aiming at the cDNA sequence of human TRPC3 in GenBank,designed and synthesized the small hairpin RNA （shRNA） and transfected it into HUVEC. The transfection efficiency was determined by laser scanning confocal microscopy,and the interference efficiency and expression of TRPC3mRNA and TRPC3 protein were determined by real time PCR and Western blot,respectively. Results demonstrated that, 1 ）Green fluorescence could be observed under the confocal microscope, showing that shTRPC3 had been transferred into HUVEC successfully. 2）shRNA decreased the expression of TRPC3mRNA and TRPC3 protein in TRPC3 shRNA group 48h after transfection（P〈0.05）. The interference efficiency was 71 % and 74 % ,respectively. The results suggested that interference plasmid of TRPC3 in HUVEC was successfully constructed.