5 789-792 转783 基础研究 4+4 特异性siRNA质粒的构建及其抑制癌细胞中mcl-1表达的研究.pdf

现代食品科技 Modern Food Science and Technology 2010, Vol.26, No.8 特异性siRNA质粒的构建及其抑制癌细胞中 mcl-1表达的研究 徐爱群，曹以诚，区镜深，张珍武 （华南理工大学生物科学与工程学院，广东广州510006 ） 摘要：本文构建了靶向mcl-l基因构建的特异性siRNA重组质粒，在细胞水平检测的其抑制效果，并筛选出能高效抑制mcl- l基因表达的siRNA重组质粒，针对mcl-1的特异性siRNA重组质粒，将其转染到人肝癌细胞HepG2 ，用Rt- PCR( 实时荧光定量PCR)方法和Western Blot方法分别检测转染前后mcl-1mRNA水平和Mcl- 1蛋白表达水平，比较对应不同位点的两个siRNA重组质粒对mcl-1的抑制效果。结果表明，Rt- PCR结果显示对应不同位点的两个siRNA重组质粒对mcl-1均可降低mcl-1 mRNA水平，最大抑制效率达70.0% ，远高于作为对照非相关组；Western-Blot结果显示转染特异性siRNA重组质粒后Mcl- 1蛋白表达受到抑制，最大抑制率为44.2% 。合理设计的特异性siRNA重组质粒可大幅度下调mcl-1 mRNA水平，能有效抑制Mcl-1蛋白表达。 关键词：肝癌；mcl-1；基因沉默；实时荧光定量PCR 文章篇号：1673-9078(2010)8-789-792 The Specific siRNA Plasmids Construction and their Inhibition Effect on Expression of mcl-1 Protein in Liver Cancer Cell XU Ai-qun, CAO Yi-cheng, OU Jing-shen, ZHANG Zhen-wu (Bioscience and Bioengineering School of South China University of Technology, Guangzhou 510006, China) Abstract: The mcl-l gene-specific siRNA plasmids were constructed and transfected into HepG2. The levels of mcl-1 mRNA and Mcl-1 protein expression were detected with the Rt-PCR (real time PCR) and WesternBlot methods, respectively. And the inhibitory effects of siRNA plasmid corresponding to two different sites on mcl-1 expression were compared. Rt-PCR results showed that both the two siRNA plasmids correspomding to two sites could reduce the levels of mcl-1 mRNA and the maximum inhibition efficiency was of 70.0%, which was much higher than that of a control group. Western-Blot results were showed that transfection of mcl-l gene-specific siRNA plasmids into HepG2. The expression of Mcl-1 protein obviously inhibit the expression of mcl-l protein with the maximum inhibition rate was 44.2%. It was concluded that the rat