小胶质细胞与神经干细胞共同培养对神经干细胞向多巴胺能神经元分化的影响

Impact of co-cultured microglia and neural stem cells via transwell on differentiation from neural stem cells into dopaminergic neurons

目的 探讨小胶质细胞与神经干细胞（NSCs）共同培养条件下对NSCs向多巴胺能神经元分化的促进作用。方法 （1）原代培养新生SD大鼠的小胶质细胞并鉴定。原代培养孕14 d SD大鼠胚胎NSCs并鉴定。（2）取鉴定后细胞分为NSCs单独培养组和小胶质细胞与NSCs共同培养组。2组细胞培养6 d后，采用细胞免疫荧光法及Western blotting实验检测多巴胺能神经元分化与成熟相关因子酪氨酸羟化酶（TH）、多巴胺转运体（DAT）及炎症因子3（Pitx3）蛋白的表达，采用PCR技术检测2组细胞TH、DAT以Pitx3基因转录水平的差异。结果 （1）新生SD大鼠小胶质细胞CD11b/c染色阳性，所得到的小胶质细胞纯度〉95%。孕14 d SD大鼠NSCs巢蛋白染色阳性。（2）细胞免疫荧光染色结果显示：与单独培养组比较，共同培养组NSCs TH、DAT及Pitx3阳性蛋白明显增多。Western blotting实验结果显示：共同培养组细胞TH、DAT以及Pitx3蛋白的表达量明显高于单独培养组，差异有统计学意义（P〈0.05）。（3）PCR实验结果显示：共同培养组TH、DAT以及Pitx3基因转录水平均明显高于单独培养组，差异有统计学意义（P〈0.05）。结论 小胶质细胞与NSCs共同培养可促进NSCs向多巴胺能神经元分化。

Objective To investigate the influence of co-culture of microglia and neural stem cells （NSCs） on differentiation from NSCs into dopaminergic neurons in vitro.Methods （1） The microglia from neonatal SD rats was purified and identified. After the NSCs were isolated from 14-day pregnant SD rats, the cells were cultured and identified. （2） The identified NSCs were randomly divided into 2 groups： simple NSCs culture group and NSCs＋microglia co-culture group. After the cells in both groups were cultured for 6 days, immunofluorescence assay and Western blotting were performed to detect the protein expression of TH, DAT and Pitx3, the factors associated with the differentiation and maturity of dopaminergic neurons. （3） PCR was used to detect the gene transcription of TH, DAT and Pitx3 in the cells from the 2 groups and differences were compared between the 2 groups.Results （1） The staining of CD11b/c in the microglia mixed cultured in the neonatal SD rats was positive, with a purity of above 95%; the staining of NSCs identified via nestin was positive in the 14-day pregnant SD rats. （2） The immunofluorescence assay showed that the amounts of positive proteins of TH, DAT and Pitx3 in the NSCs＋microglia co-culture group were significantly larger than in the simple NSCs culture group （P〈0.05）. The Western blotting showed that the protein expression levels of TH, DAT and Pitx3 in the NSCs＋microglia co-culture group were significantly higher than in the simple NSCs culture group （P〈0.05）. （3） The PCR detection showed that the gene transcription levels of TH, DAT and Pitx3 in the NSCs＋microglia co-culture group were significantly higher than in the simple NSCs culture group （P〈0.05）.Conclusion Co-culture of NSCs and microglia via Transwell may promote differentiation from NSCs into dopaminergic neurons.