核受体相关因子1表达下调对多巴胺能细胞酪氨酸羟化酶和神经突起生长的影响

Effects of Nurr1 down-regulation on the expression of tyrosine hydroxylase and neurite extension in dopaminergic cells

将合成的核受体相关因子1(nuclear receptor-related factor 1,Nurrl)特异性短发夹寡核苷酸(small-hairpin RNA,shRNA)序列插入真核表达载体pSilenCircle(pSC),构建Nurrl基因特异性shRNA真核表达载体,转染体外培养多巴胺能神经前体细胞系MN9D,分别采用实时荧光定量PCR和Western blot方法检测其对MN9D细胞内源Nurrl的干扰作用及其对酪氨酸羟化酶(tyrosine hydroxylase,TH)表达的影响,并在倒置显微镜下观察MN9D细胞神经突起生长的情况,探讨Nurrl shRNA表达载体对多巴胺能细胞表型标记物TH和以神经突起延长为特征的细胞成熟的影响。结果表明,脂质体组细胞和转染阴性对照质粒的MN9D细胞内Nurrl、TH的表达正常,而转染Nurrl shRNA真核表达载体(pSC-N1和pSC-N2)的MN9D细胞内Nurrl和TH的mRNA水平明显降低,Nurrl mRNA的下降率分别为62．3%和45．65%,TH mRNA的下降率分别为76．3%和62．6%。同时Nurrl和TH蛋白的表达亦明显下调,Nurrl蛋白的下降率分别为57．4%和72．0%,TH蛋白的下降率分别为79．1%和70．1%。另外,转染Nurrl shRNA真核表达质粒的MN9D细胞神经突起延长有所减少,但是与正常细胞无明显差异。结果提示：Nurrl shRNA真核表达载体能显著下调MN9D细胞内源Nurrl和TH mRNA和蛋白的表达,同时可能对MN9D细胞的神经突起延长有一定的抑制作用。Nurrl shRNA表达载体的成功构建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。

In the experiment, we designed and synthesized two siRNAs based on the sequence of nuclear receptor-related factor 1 （Nurrl） mRNA. They were separately subcloned into the plasmid of pSilenCircle （pSC） containing U6 promoter. The pSC-Nurrl vectors （pSC-N1 and pSC-N2） specific to Nurrl gene and the negative control vector of short-hairpin RNA （shRNA） eukaryotic expression vector were constructed. We cultured the dopaminergic cell line MN9D and the verified vectors were transfected with LipofectamineTM 2000 in vitro. The positive cell clones transfected with pSC were obtained after being screened with 500μg/ml G418. After that, the silencing effects of Nurrl and TH mRNA or protein were detected by real time RT-PCR and Western blot. The neurite extension of MN9D cells was observed and photographed by inverted microscope. The results showed that Nurrl mRNA expression in MN9D cells was specifically down-regulated by the vectors of pSC-N1 and pSC-N2, and the silencing effects were 62.3% and 45.6%, respectively. The dopaminergic phenotype of TH mRNA was also suppressed significantly and the silencing effects were 76.3% and 62.6%, respectively. Meanwhile, the expressions of Nurrl and TH proteins were also significantly suppressed, and the silencing effects of Nurrl and TH protein were 57.4%, 72.0% and 79.1%, 70.1% respectively. The negative control and liposome groups had no effect on the two genes. In conclusion, Nurrl shRNA expressing vectors can inhibit the expressions of Nurrl and TH mRNA or protein in MN9D cells, and Nurrl might play a role in neurite extension of MN9D cells. Nurrl shRNA expressing vector may provide a novel applicable strategy for the study on the function of the genes associated with Parkinson＇s disease and the development of dopaminergic neuron.