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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 study the inhibitory effect of siRNA on rat cardiac cystein sulfonate decarboxylase (CSD). Methods Four siRNAs and one negative control siRNA expression plasmid were designed and constructed according to the cloned rat cardiac CSD gene sequence (EU786150). The constructed recombinant plasmids siRNA1 #, siRNA2 #, siRNA3 # The transfected H9c2 cells were transfected with siRNA4 # and negative control plasmids respectively. The transfection efficiency was observed by fluorescence microscopy. The proliferation of H9c2 cells was detected by MTT assay. The mRNA and protein expression of CSD were detected by real-time fluorescence quantitative PCR and Western-blot. Results Compared with negative control group and untransfected group, siRNA1 # recombinant plasmid significantly inhibited the expression of CSD mRNA and protein in H9c2 cells (P <0.05), while siRNA4 # plasmid (P> 0.05). The results of MTT assay showed that the proliferation of H9c2 cells transfected with recombinant plasmid siRNA1 # and siRNA2 # was significantly inhibited at 72 h and 48 h, respectively. Compared with negative control group, untransfected group Compared with the difference was significant (P <0.05). Conclusion siRNA1 # recombinant plasmid can effectively inhibit the expression of CSD gene and significantly inhibit the proliferation of H9c2 cells, which lays the foundation for studying the gene of CSD and revealing the role of taurine in cardiomyocyte metabolism. [Journal of Nutrition, 2012,34 (4): 317-321]