小鼠胚胎大脑皮质与中脑神经干细胞培养与分化比较的研究

目的比较小鼠胚胎脑皮质与中脑来源的神经干细胞在培养与分化方面的差异，为更好的研究和利用神经干细胞提供实验依据。方法无菌条件下分别分离鼠胚大脑皮质与腹侧中脑，经胰酶消化及机械吹打成单细胞后接种于含有B27、bFGF的DMEM／F12培养基，培养扩增；倒置显微镜观察比较生长状况；机械方法传代；10％血清接种分化；免疫荧光细胞化学方法染色鉴定神经干细胞及其子代细胞的分化方向，并作比较。结果小鼠胚胎脑皮质与中脑均存在神经干细胞，其免疫细胞化学鉴定均呈Nestin阳性，并都能分化为神经元和胶质细胞；但皮质部位所含神经干细胞明显多于中脑，也更宜成球；有血清条件下分化，皮质神经干细胞未见分化为酪氨酸羟化酶（TH）阳性神经元；中脑神经干细胞则可少量分化为TH阳性神经元结论同样条件下皮质神经干细胞比中脑神经干细胞更易增殖与培养，两者在有血清条件下分化为TH阳性神经元能力方面有差异。

A PRELIMINARY RESEARCH IN CULTIVATION AND DIFFERENTIATION OF CORTICAL AND MESENCEPHALIC NEURAL STEM CELLS IN VITRO

Objective To explore the differences of cultivation and differentiation between cortical neural stem cells(C-NSCs) and mesencephalic neural stem cells(M-NSCs). Methods We dissociated embryonic cortex and ventral mesencephalon respectively,and single cell suspensions were achieved by trypsin digestion and mechanical dissociation in sterile condition.These cells were planted into DMEM/F12 culture medium with B27,bFCF,and then were passaged by mechanical methods and induced to differentiation in DMEM/F12 medium with 10%FBS(without growth factors).For examining the features of C-NSCs and M-NSCs,reverse phasecontrast microscopy,immunocytochemical techniques were applied to detect nestin,NSE,GFAP antigens and the specific antigen(TH) of neurons differentiated from the two kinds of NSCs.We observed and compared the difference of the two kinds of NSCs. Results The NSCs existed both in cortex and ventral mesencephalon of embryonic mice,which expressed nestin and possessed the ability of self-renewing and multipotent, but the density of NSCs in cortex was higher than that in ventral mesencephalon.There were some TH positive cells differentiated from M-NSCs but none came from C-NSCs.Conclusion The proliferative activity of C-NSCs is higher than that of M-NSCs,but more number M-NSCs differentiated into dopaminergic neurons in serum medium.