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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
吴云成1, 蔡友庆2, 赵永波1,*, 费 俭2
1上海市第一人民医院神经内科、临床神经生化研究室，上海 200080; 2中国科学院上海生命科学研究院生物化学与细胞生物学研究所，上海 200031
摘 要：将合成的核受体相关因子1 (nuclear receptor related factor 1, Nurr1)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)，构建出特异性Nurr1基因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Nurr1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Nurr1 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Nurr1、TH的表达正常，而转染Nurr1 shRNA真核表达载体(pSC-N1和pSC-N2)的MN9D细胞内Nurr1和TH的mRNA水平明显降低，Nurr1的下降率分别为62.3%和45.6%，TH 的下降率分别为76.3%和62.6%。同时Nurr1和TH蛋白的表达亦明显下调，Nurr1的下降率分别为57.4％和72.0％，TH 的下降率分别为79.1％和70.1％。另外，转染Nurr1 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：Nurr1 shRNA真核表达载体能显著下调MN9D细胞内源Nurr1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；Nurr1 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。
关键词：核受体相关因子1；酪氨酸羟化酶；RNA干扰; 帕金森病；发育；神经突起
Effects
of Nurr1 down-regulation on the expression of tyrosine hydroxylase and neurite
extension in dopaminergic cells
WU Yun-Cheng1,
CAI You-Qing2, ZHAO Yong-Bo1,*, FEI Juan2
1Department of Neurology, Shanghai First
People’s Hospital, Shanghai 200080, China; 2Institute
of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, China
Abstract: In the experiment, we designed and
synthesized two siRNAs based on the sequence of nuclear receptor-related factor
1 (Nurr1) mRNA. They were separately subcloned into the plasmid of pSilenCircle
(pSC) containing U6 promoter. The pSC-Nurr1 vectors (pSC-N1 and pSC-N2) specific
to Nurr1 gene and the negative control vector of short-hairpin RNA 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 500 µg/ml G418. After that, the
silencing effects of Nurr1 and TH mRNA or protein were detected by Real Time
RT-PCR and Western blot. The neurite extension of MN9D cells were observed and
photographed by inverted microscope. The results showed that Nurr1 mRNA
expression in MN9D cells was specifically down-regulated by the vectors of
pSC-N1 and pSC-N2, the silencing effect is 62.3% and 45.6% respectively. The
dopaminergic phenotype of TH mRNA is also suppressed significantly and the
silencing effect is 76.3% and 62.6% respectively. Meanwhile, the expression of
the protein of Nurr1 and TH was significantly suppressed too, the silencing effect of Nurr1 and TH protein is 57.4%, 72.0% and 79.1％, 70.1％ respectively. The negative control and liposome groups
have no effect on the two genes. In conclusion, Nurr1 shRNA expressing vectors
can inhibit the expression of Nurr1 and TH mRNA or protein in MN9D cells, and Nurr1
might play a role in neutite extension of MN9D cells. Nurr1 shRNA expression
vector may provide a novel applicable strategy for the study on the function of
the genes associated with Parkinson disease and the development of dopaminergic
neuron.
Key words: Nurr1 nuclear receptor, Tyrosine 3-monooxygenase;
RNA interference; Parkinson disease; development; neurites