外源性Nurr1基因过表达对SK-N-SH细胞分化作用的研究

为研究外源性Nurr1基因过表达对转染细胞的作用,探讨Nurr1基因调控多巴胺能神经元分化的机制。本研究应用:(1)pBK- RSV -Nurr1质粒经脂质体法转染SK- N- SH细胞,G418筛选;用RT PCR和免疫细胞化学方法检测基因转染细胞Nurr1mRNA及Nurr1蛋白的表达; (2)all- trans- RA、9- cis- RA、forskolin诱导基因转染细胞,RT PCR及免疫荧光细胞化学方法检测基因转染细胞MAP2 和TH的表达。结果显示: (1)经RT -PCR检测,基因转染细胞表达Nurr1mRNA;免疫细胞化学染色结果显示基因转染细胞表达Nurr1蛋白,说明外源性Nurr1基因在转染细胞内过表达;基因转染细胞形态与正常细胞相比没有明显变化,生长速度略有降低并表达成熟神经元的特异性标识物MAP2,但不表达多巴胺能神经元的特异性标识物TH。(2)尽管RA、forskolin诱导可促进Nurr1基因转染的SK- N- SH细胞进一步向成熟神经元分化,但不能诱导基因转染细胞表达TH。结论:单纯的Nurr1过表达可以促进SK- N- SH细胞向成熟神经元方向分化,但不足以激活TH基因转录。TH基因的转录激活还需要除Nurr1以外的其它细胞(或神经元)的环境或因子的共同作用。

EFFECT OF EXOGENOUS Nurr1 GENE OVEREXPRESSION FROM CULTURED SK-N-SH CELLSON CELLULAR DIFFERENTIATION

To establish a SK-N-SH cell model overexpressing the gene of Nurr1 and to investigate whether the overexpression of exogenous Nurr1 can facilitate the cellular differentiation. 1.The SK-N-SH cells were transfected with the pBK-RSV-Nurr1 plasmid by LipofectAMINE and selected by G418. The expression of Nurr1 was detected by RT-PCR and immunocytochemistry. 2.The Nurr1-overexpressing cells were treated with all-trans-RA, 9-cis-RA and forskolin. Then the expression of MAP_2 and TH were detected by RT-PCR and immunocytochemistry. The results showed that: (1) The Nurr1-overexpressing SK-N-SH cells showed no obvious difference in morphology from the parental cells, with exceptions that they showed slightly slow growth rate. RT-PCR analysis and immunocytochemistry staining showed that the Nurr1-overexpressing SK-N-SH cells expressed MAP_2, a special maker for differentiated neurons, a phenomenon not seen in the parent SK-N-SH cells. However, the Nurr1-overexpression cells did not express TH, a marker for dopaminergic neurons. (2) Although the transgenic cells showed a more differentiated neuronal morphology and expressed MAP_2 after treatment with all-trans-RA, 9-cis-RA and Forskolin, none of these factors induced TH expression in Nurr1-overexpressing SK-N-SH cells. Conclusion:The overexpression of Nurr1 in SK-N-SH cells promotes differneciation. But Nurr1 alone is not sufficient to induce dopaminergic phenotypes. The activation of TH gene probably requires the effect of specific cellular (or neuronal) environments or cofactors except Nurr1 overexpression.