核受体相关因子1 shRNA表达载体的构建及其对多巴胺能细胞内源基因的干扰作用

目的 构建小鼠核受体相关因子1（Nurr1）shRNA的真核表达载体,并检测其对多巴胺能细胞内源基因的干扰作用.方法 构建两个针对Nurr1的RNA干扰重组表达载体,分别命名为pSC-N1和pSC-N2.经测序确认后,转染多巴胺能神经前体细胞系MN9D细胞株,以实时荧光定量PCR以及Western blot方法检测转染后Nurr1 mRNA和蛋白的表达.结果 测序鉴定证实合成的siRNA基因序列正确并已被准确克隆入pSilenCircle载体.pSC-N1和pSC-N2可特异性抑制MN9D细胞中Nurr1 mRNA的表达,下降率分别为62.3%和45.6%;Nurr1蛋白的表达亦明显下调,其下降率分别为57.4%和72.0%,而对照质粒对其无抑制作用.结论 成功构建了能特异性抑制多巴胺能细胞内源Nurr1表达的shRNA表达载体,为多巴胺能神经元发育以及帕金森病相关基因的功能研究奠定了基础.

Construction of vectors generating Nurr1 shRNA and their silencing effect in cultured dopaminergic cell lines

Objective To construct the nuclear receptor-related factor 1 ( Nurr1) specific short-hairpin RNA (shRNA) expressing vectors and study their effects on the expression of endogenous genes in MN9D dopaminergic cell lines. Methods Two RNAi recombinant plasmids (named pSC-N1 and pSC-N2) targeting at Nurrl gene were constructed. The purified plasmids were identified by DNA sequencing. After being transfected into MN9D dopaminergic cell lines with Lipofectamine?2000, the silencing effects of Nurrl shRNA expressing vectors on the expression of Nurrl mRNA and protein in MN9D cells were detected using real time fluorescence quantitative PCR and Western blot Results DNA sequencing confirmed that the Nurrl shRNA expressing vectors were constructed successfully. Nurrl mRNA expression in MN9D cells was specifically suppressed after the transfection of pSC-Nl and pSC-N2 by 62.3% and 45.6% respectively. Moreover, the expression of Nurrl protein was significantly suppressed by 57.4% and 72. 0% respectively. The control vectors had no silencing effect Conclusions The vectors pSC-Nl and pSC-N2 have been successfully constructed and can specifically suppress the expression of Nurrl mRNA and protein. Nurrl specific shRNA expressing vector may provide a novel applicable strategy for the study of the function of the genes associated with Parkinson disease and the development of dopaminergic neuron.