小鼠中脑神经干细胞体外培养方法的研究

目的改进小鼠中脑神经干细胞(NSCs)分离培养方法。方法分别采用机械法和酶消化法分离出生1～3d的小鼠中脑组织,在含体积分数为2%B27及20ng/mL碱性成纤维细胞生长因子(bFGF)的DMEM/F12培养基中进行原代和传代培养。采用巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)、及神经元特异性烯醇化酶(NSE)免疫细胞化学染色方法鉴定NSCs和分化细胞。结果在含bFGF及B27的DMEM/F12培养基中,可形成悬浮生长Nestin呈阳性的神经球。诱导分化的细胞可见GFAP或NSE免疫反应阳性细胞。机械法较酶消化法可获得较多的NSCs。结论在DMEM/F12培养基中添加2%B27及20ng/mL的bFGF可成功获得中脑NSCs,机械法为一种简便而有效的NSCs分离方法。

Research of the mouse mesencephalic neural stem cells cultured in vitro

Objective To improve the separation and cultivation methods of the mouse mesencephalic neural stem cells.Methods Using mechanical method and enzyme digestion methods,the mouse mesencephalic tissues were separated after birth from 1 to 3 days.After being cultured or subcultured in DMEM/F12 culture medium which had a volume fraction of 2% B27 and 20 ng/mL bFGF,immunocytochemical staining method such as Nestin,glial fibrillary acidic protein(GFAP),and neuron specific enolase(NSE) were used to identify neural stem cells and differentiated cells.Results In the DMEM/F12 culture medium which contain bFGF and B27,Nestin positive neurosphere was suspension growing.The GFAP or NSE immunoreactivity positive cells could be found in the cells after induction of differentiation.Compared with enzyme digestion method,the mechanical method could obtain more neural stem cells.Conclusion Adding 2% B27 and 20 ng/mL bFGF into DMEM/F12 culture medium can obtain mesencephalic neural stem cells successfully.The mechanical method is a more simple and more effective method in neural stem cells separation.