人小龄胚胎神经干细胞的分离培养、扩增及鉴定

目的 探索人胚胎神经干细胞的体外分离培养条件，从而在体外大量扩增神经干细胞；并观察神经干细胞增殖、分化的特点。方法 从人胚胎脑分离神经干细胞，部分细胞冻存，另一部分细胞进行体外培养。采用无血清培养液，加入表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)刺激细胞增殖，进行体外扩增、传代培养。采用免疫荧光法鉴定神经干细胞和分化的神经元及胶质细胞。结果 从人胚胎脑分离的细胞在含有EGF和bFGF的无血清培养液中能形成大量的神经干细胞球，这些神经干细胞球可在体外扩增及传代培养。免疫荧光法鉴定神经干细胞球中大部分为神经上皮干细胞蛋白(nestin)表达阳性细胞。贴壁后可以分化出神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)表达阳性的细胞。经冻存后的胎脑细胞也能培养出具有同样特征的神经干细胞。结论 在含有EGF和bFGF的无血清培养液中，从人胚胎脑能分离培养出神经干细胞，并能在体外大量扩增。这为人类神经干细胞的进一步研究和应用提供了材料。

Isolation,cultivation,expansion and identification of human fetal neural stem cells in vitro

Objective To explore the methods of isolation, cultivation and expansion of human embryonic neural stem cells, and to observe the characteristics of proliferation and differentiation of neural stem cells in vitro. Methods The neural stem cells were isolated from human embryo, some cells were frozen and the others were cultured. A serum free medium containing EGF and bFGF was used to cultured and expanded neural stem cells. Immunocytofluorescence was used to identifying the neural stem cells and their potentiality of differentiation. Results The neurospheres could be conformed in serum-free medium from cells that was isolated from human fetal brain, which could be expanded and passaged in the serum free medium with EGF and bFGF. Nestin positive cells can be found in the neurospheres. After attachment they can differentiate into GFAP and NSE positive cells. The same phenotype cells could also be harvested from those frozen cells by reculturing. Conclusion The neural stem cells isolated from human fetal brain can be cultured and expanded in vitro. It can be used to further study on the human neural stem cells.