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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。
To investigate the effect of exogenous Nurr1 gene overexpression on transfected cells and to explore the mechanism of Nurr1 gene regulating dopaminergic neuron differentiation. In this study, (1) pBK-RSV-Nurr1 plasmid was transfected into SK-N-SH cells by lipofectamine and then screened by G418. The expression of Nurr1 mRNA and Nurr1 protein were detected by RT-PCR and immunocytochemistry. (2) The transfected cells were induced by all-trans-RA, 9-cis-RA and forskolin. The expression of MAP2 and TH were detected by RT-PCR and immunofluorescence cytochemistry. The results showed that: (1) Nurr1 mRNA was expressed in transfected cells by RT-PCR; Nurr1 protein was expressed in transfected cells by immunocytochemical staining, indicating that Nurr1 gene was overexpressed in transfected cells; Cell morphology did not change significantly compared with normal cells, with a slight decrease in growth rate and expression of MAP2, a specific marker of mature neurons, but not of TH, a specific marker of dopaminergic neurons. (2) Although RA and forskolin induce further differentiation of SK-N-SH cells transfected with Nurr1 gene into mature neurons, they can not induce TH-expressing cells. CONCLUSION: Overexpression of Nurr1 can promote the differentiation of SK-N-SH cells into mature neurons but not enough to activate TH gene transcription. The transcriptional activation of the TH gene also requires the co-action of the environment or factors of other cells (or neurons) than Nurr1.