李坤 丁宁 李琳

    [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(pLNCX-EGFP-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。

    [关键词] 人核糖核酸酶抑制因子；siRNA；鉴定

    [中图分类号] R78 [文献标识码] A [文章编号] 1673-7210(2014)09(a)-0008-04

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

    LI Kun1 DING Ning1 LI Lin2

    1.Department of Clinical Laboratory Medicine， Medical College of Dalian University， Liaoning Province， Dalian 116021， China； 2.Department of Clinical Laboratory， Maternal and Child Care Service Centre of Dalian， Liaoning Province， Dalian 116021， China

    [Abstract] 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) ......