小鼠胚胎中脑神经干细胞分离、培养及分化

目的:探讨小鼠胚胎中脑分离神经干细胞的体外培养方法,以获取高纯度的神经干细胞,为神经干细胞的深入研究提供试验材料。方法:无菌条件下分离孕12~13 d小鼠胚胎中脑曲,制成单细胞悬液,碱性成纤维生长因子(bFGF)和B27存在的培养基中培养扩增,通过免疫细胞化学染色鉴定神经干细胞及子代细胞的分化方向,流式细胞术检测TH阳性神经元比例。结果:培养的部分细胞体外分裂增殖,同时表达神经干细胞特异性抗原nestin,并向神经细胞和胶质细胞分化并经流式细胞仪检测自然分化为多巴胺能神经元的比例为3.25%。结论:小鼠胚脑中脑存在具有多分化潜能的神经干细胞,它们能在体外稳定培养、传代和分化。

Isolation Cultivation and Differentiation of Mesencephalic Neural Stem Cells of Embryo Mice-Derived

Objective: To explore the culture methods of Mesencephalic neural stem cells from embryonic mice and to obtainhighly purified neural stem cells for further investigation.Methods: Ventral mesencephalons of embryonic mices(E12-13) were dissociated and single cell suspensions were achieved in sterile condtions.Then the cells were cultured in DMEM/F12 supplemented with bFGF and B27.Immunocylochemical techniques were used to odentify stem cells and their progeny.FCM detects the percentage of TH-ir in all the cells.Result:In the serunrtree medium containing bFGF and B27,the cells from embryonic mice divided and expanded in vitro,in which nestin antigen was expressed.In the containing serum medium but neither bFGF nor B27,the cells differentiated into neurons and glia.The proportions of M-NSCs differentiation into DA ergic neurons were about 3.25%,Conclusion:Neural stem cell from the ventral mesencephal can be cultured,passaged and differentiated in vitro.They are the ideal cell sources for cell transplantation therapy.