Nurr1对小胶质细胞联合神经干细胞共培养促进神经干细胞向多巴胺神经元分化作用研究

Effect of nuclear receptor-related factor 1 on differentiation of neural stem cells into dopaminergic neurons in the co-culture system of neural stem cells and microglial cells

目的探讨联合过表达核受体相关因子1(Nurr1)基因的小胶质细胞(MG)和神经干细胞(NSC)共培养对神经干细胞向多巴胺神经元分化的影响。方法原代培养SD大鼠神经干细胞和小胶质细胞,并过表达Nurr1基因。CCK-8法检测Nurr1过表达对神经干细胞以及小胶质细胞活率的影响。Transwell系统共培养神经干细胞和小胶质细胞,实验分为NSC组、NSC+MG组和N(NSC+MG)组。ELISA检测共培养后第3天、第6天和第9天各组脑源性神经营养因子(BDNF)、血小板源性神经营养因子(PDNF)和胶质细胞源性神经营养因子(GDNF)表达变化;RT-PCR和Western Blot检测各组第9天酪氨酸羟化酶(TH)、多巴胺转运蛋白(DAT)DAT和Nurr1的表达变化;细胞免疫荧光鉴定神经干细胞的分化,并对TH和DAT阳性细胞计数,计算各组神经干细胞向多巴胺神经元的分化效率。结果原代培养小胶质细胞以及神经干细胞并成功过表达Nurr1基因。CCK-8法检测结果表明,Nurr1过表达对神经干细胞以及小胶质细胞活率无明显影响。ELISA检测结果表明,N(NSC+MG)组在不同时间点神经营养因子(BDNF、PDNF和GDNF)表达量明显高于其他各组(P<0.05)。RT-PCR和Westen Blot检测结果表明,N(NSC+MG)组TH、DAT和Nurr1的表达水平明显高于其他各组(P<0.05)。细胞免疫荧光鉴定结果表明,N(NSC+MG)组TH阳性细胞率明显高于其他各组(P<0.05)。结论Nurr1基因可促进神经干细胞和小胶质细胞共培养系统神经营养因子的分泌。过表达Nurr1基因的神经干细胞和小胶质细胞共培养可促进神经干细胞向多巴胺神经元的分化。

Objective To investigate the effect of co-culture of microglial cells （MGs） and neural stem cells （NSCs） with overexpres- sion of nuclear receptor-related factor 1 （ Nurrl ） gene on the differentiation of NSCs into dopaminergie neurons. Methods Primary NSCs and MGs were cultured, and the overexpression of Nurrl gene was performed. Cell Counting Kit-8 （ CCK-8 ） assay was used to investigate the influence of Nurrl overexpression on the viability of NSCs and MGs. The Transwell co-culture system was used for the co-culture of NSCs and MGs, and the cells were divided into NSC group, NSC ＋ MG group, and N（ NSC ＋ MG） group. ELISA was used to measure the expression of brain-derived neurotrophie factor （BDNF）, platelet-derived neurotrophic factor （PDNF） , and glial cell-derived neurotrophic factor （GDNF） on days 3, 6, and 9 of co-culture ; RT-PCR and Western blot were used to measure the ex- pression of tyrosine hydroxylase （TH）, dopamine transporter （DAT）, and Nurrl on day 9; cell immunofluorescence was used to deter- mine cell differentiation and the numbers of TH~ and DAT ~ cells, and the efficiency of the differentiation of NSCs into dopaminergic neurons was calculated. Results Primary NSCs and MGs were successfully cultured, and the overexpression of Nurrl gene was suc- cessfully performed. The CCK-8 assay showed that Nurrl overexpression had no significant influence in the viability of NSCs and MGs.The resuhs of ELISA showed that the N（ NSC ＋ MG） group had significantly higher expression of BDNF, PDNF, and GDNF at different time points than the other two groups （P 〈0.05）. RT-PCR and Western blot showed that the N（NSC ＋ MG） group had significantly higher expression of TH, DAT, and Nurrl than the other two groups （P 〈 0.05）. Cell immunofluorescence showed that the N（ NSC ＋ MG） group had a significantly higher percentage of TH ＋ cells than the other two groups （P 〈 0.05 ）. Conclusions Nurrl can pro- mote the secretion of neurotrophic factors in the co-culture system of NSCs and MGs, and overexpression of Nurrl can promote the dif- ferentiation of NSCs into dopaminergic neurons in this co-culture system.