shRNA真核表达质粒介导的RNA干扰对人结肠癌LoVo细胞mTOR基因表达的抑制作用

目的探讨shRNA真核表达质粒介导的RNA干扰技术对人结肠癌LoVo细胞哺乳动物雷帕霉素靶蛋白（mTOR）基因表达的抑制作用。方法合成编码shRNA的特异性DNA序列，连接到质粒pGenesil-1，采用脂质体Lipofectamine^TM 2000转染LoVo细胞，G418筛选4周。根据转染质粒的不同，实验分为3组：A组（正常对照组）为未转染的正常培养LoVo细胞，B组（阴性对照组）为转染pGenesil-1空载体质粒，C组（阳性实验组）为转染质粒pGenesil-1-mTOR。采用逆转录聚合酶链反应（RT—PCR）法检测LoVo细胞mTOR基因mRNA表达，免疫印迹（Western blot）法检测mTOR蛋白表达。结果双酶切产物琼脂糖凝胶电泳显示，重组质粒pGenesil-1-mTOR和空载体质粒pGenesil-1分别切出一条410、350bp大小的片段。G418筛选的转染细胞在倒置荧光照相显微镜下可观察到绿色荧光。C组LoVo细胞mTOR基因mRNA表达水平明显低于A组（P〈0.05），表达抑制率为73．0％。C组LoVo细胞mTOR蛋白表达水平明显低于A组（P〈0．05），表达抑制率为72．5％。结论shRNA真核表达质粒介导的RNA干扰可明显抑制人结肠癌LoVo细胞mTOR基因表达。

Inhibition Effect of RNA Interference with Eukaryotic Expression Plasmids Containing shRNA on mTOR Gene Expression in Human Colon Cancer LoVo Cell Line

Objective To explore inhibitory effect of RNA interference with eukaryotic expression plasmid containing shRNA on expression of mTOR gene in human colon cell line LoVo.Methods Specific DNA sequences encoding shRNA were synthesized and inserted into plasmid pGenesil-1.Then these recombinant plasmids were transfected into LoVo cells with LipofectamineTM 2000 and the transfected cells were selected for 4 weeks by using G418.There were three groups in this study: group A the normal cultured LoVo cells untransfe...