siRNA表达载体对大鼠心肌H9c2细胞牛磺酸合成限速酶-CSD基因表达的抑制作用

THE INHIBITORY EFFECTS OF siRNA EXPRESSION VECTOR ON THE TAURINE BIOSYNTHETIC RATE-LIMITING ENZYME-CSD GENE OF RAT H9C2 CARDIAC MYOBLASTS

目的研究siRNA对大鼠心肌半胱亚磺酸脱羧酶(CSD)的抑制作用。方法根据已克隆的大鼠心肌CSD基因序列(EU786150)设计并构建了4个siRNAs及1个阴性对照siRNA表达质粒,利用脂质体LipofectamineTM2000将构建好的重组质粒siRNA1#、siRNA2#、siRNA3#、siRNA4#及阴性对照质粒分别转染H9c2细胞,应用荧光显微镜观察转染效率、四唑盐(MTT)法检测H9c2细胞增殖情况、实时荧光定量PCR和Western-blot检测CSD mRNA和蛋白表达。结果荧光显微镜观察细胞转染效率达70%左右;与阴性对照组、未转染组比较,siRNA1#重组质粒显著抑制了H9c2细胞中CSD mRNA和蛋白的表达(P<0.05),而siRNA4#质粒对CSD表达无明显抑制效应(P>0.05);MTT检测结果表明转染重组质粒siRNA1#、siRNA2#进入H9c2细胞48 h、72 h后细胞增殖明显受抑制,与阴性对照组、未转染组相比差异均达到显著水平(P<0.05)。结论 siRNA1#重组质粒能有效抑制CSD基因的表达,且显著抑制H9c2细胞增殖,为研究CSD基因及揭示牛磺酸在心肌细胞代谢中的作用奠定基础。[营养学报,2012,34(4):317-321]

Objective To investigate the inhibitory effects of RNAi（RNA interference） on CSD expression in rat H9c2 cells.Methods Four siRNAs expression vectors and one negative control siRNA expression vector were constructed to be targeted directly at cysteine sulfinate deacrboxylase（CSD） gene（Biomics Biotechonolgies,China）.Then the recombinant plasmids siRNA1#、siRNA2#、siRNA3#、siRNA4# and siRNA-Neg were transfected into H9c2 cells with liposomes Lipofectamine 2000.After transfection,the transfection efficiency was observed under fluorescent microscopy.CSD mRNA and protein expression were examined using RT-PCR and Western blot.MTT assay was performed to detect the state of cell proliferation.Results The transfection efficiency was about 70%.Recombinant plasmid siRNA1# significantly decreased the expression of CSD at gene and protein levels（P0.05）.However,the recombinant plasmid siRNA4# had no significant inhibition effect on CSD expression（P0.05）.Cell proliferation in the transfected cells was inhibited obviously by siRNA1# and siRNA2# plasmid compared with untransfected cells and negative control cells at 48 h,72 h（P0.05）.Conclusion The siRNA1# recombinant plasmid could effectively inhibit the expression of CSD in rat H9c2 cells and proliferation of H9c2 cells,which will benefit the further study on the function of CSD and taurine in cardiocytes metabolism.