siRNA干扰人核糖核酸酶抑制因子在HeLa细胞中的表达鉴定  

Identification of the effect of siRNA plasmid targeted combination hRI expressed in HeLa cell

作　　者：李坤[1] 丁宁[1] 李琳 

机构地区：[1]大连大学医学院检验系,辽宁大连116021 [2]大连市妇幼保健院检验科,辽宁大连116021

出　　处：《中国医药导报》2014年第25期8-10,25,共4页China Medical Herald

基　　金：辽宁省教育厅科研课题(编号L2011219)

摘　　要：目的鉴定已构建的干扰人核糖核酸酶抑制因子(hRI)的siRNA表达载体pKD-dsRI的干扰效果。方法用脂质体法将所构建的pKD-dsRI与充当报告基因的重组绿色荧光蛋白融合hRI的逆转录病毒载体(pLNCXEGFP-C1-hri)共转染到人宫颈癌HeLa细胞中。实验设4组:空白细胞组(未转染组)、pLNCX-EGFP-C1-hri转染组(对照组1)、pKD+pLNCX-EGFP-C1-hri共转染组(对照组2)及pKD-dsRI+pLNCX-EGFP-C1-hri共转染组(干扰组)。采用RT-PCR和Western blotting检测egfp-hri基因在转录后基因水平和蛋白质水平的表达。结果干扰组egfp-hri基因的mRNA转录水平和蛋白表达水平较对照组1与对照组2分别下降了91%、85%和83%、81%(P<0.05)。结论已成功构建了针对hRI的siRNA表达载体。Objective To identify the silencing effect on human HeLa cells of a siRNA plasmid pKD-dsRI targeting the gene of human ribonuclease inhibitor （hRI）, which is capable of expression in mammalian cells. Methods The vector of pKD-dsRI was co-transfected into HeLa cells with the reporter plasmid of pLNCX-EGFP-C1-hri （transfection group）, using the cells transfected with the reporter plasmid of pLNCX-EGFP-C1-hri （control group 1）, empty plasmid of pKD together with pLNCX-EGFP-C1-hri （control group 2）, and those untransfected （no transfection group） as controls. The silencing effect was determined by RT-PCR, and the expression level of egfp-hri protein was determined by Western blotting respectively. Results The transcription level of the egfp-hri fusion gene in cells co-transfected with pKD-dsRI and reporter plasmid decreased by 91%and 85%, while the expression level of the egfp-hri fusion gene decreased by 83% and 81%, as compared with control group 1 and control group 2 respectively （each P〈0.05）. Conclusion The plasmid of siRNA targeting hRI is successfully constructed and the protein of hRI can be inhibited in cytomatrix of HeLa cells correctly.

关 键 词：人核糖核酸酶抑制因子 SIRNA 鉴定 

分 类 号：R78[医药卫生—口腔医学]