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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 mBNA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人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。
Objective To construct the eukaryotic expression vector of mouse nuclear receptor - related factor 1 (Nurr1) shRNA and detect its interference with the endogenous gene of dopaminergic cells. Methods Two RNAi recombinant expression vectors targeting Nurr1 were constructed and named pSC-N1 and pSC-N2. After sequencing, the cells were transfected with MN9D cells, and the expression of Nurr1 mBNA and protein was detected by real-time fluorescence quantitative PCR and Western blot. Results Sequencing and identification confirmed that the synthetic siRNA gene sequence was correct and had been cloned correctly in human pSilenCircle vector. pSC-N1 and pSC-N2 can specifically inhibit the expression of Nurr1 mRNA in MN9D cells, the decreasing rates were 62.3% and 45.6% respectively; the expression of Nurr1 protein was also significantly down-regulated, with the decreasing rates of 57.4% And 72.0% respectively, whereas the control plasmid did not inhibit it. Conclusion The shRNA expression vector which can specifically inhibit the expression of endogenous Nurr1 in dopaminergic cells was successfully constructed and laid the foundation for the development of dopaminergic neurons and the function of genes related to Parkinson’s disease.