大鼠胎脑神经干细胞的培养分化及鉴定的实验研究

Experimental study of cultivation,differentiation and identification in fetal rats neural stem cells

目的建立大鼠胎脑皮质神经干细胞的培养、扩增、诱导分化及鉴定的方法。方法选取孕14d胎鼠的大脑皮质作为细胞来源,在无血清培养基中添加B27、碱性成纤维细胞因子、表皮生长因子,建立胚胎神经干细胞的体外培养体系,用5%的胎牛血清诱导神经干细胞分化,用免疫荧光染色技术进行对神经干细胞及诱导分化细胞的鉴定。结果原代培养的神经干细胞折光性强,培养第2d开始形成细胞集落,第3d形成大的神经球。3～5代传代的细胞生长稳定。经胎牛血清诱导后第3d开始,神经球开始向周边发出突起,悬浮的细胞开始贴壁生长。原代及传代培养的神经球表达Nestin阳性,分化细胞分别表达NSE、GFAP和GALC阳性。结论应用无血清培养技术可在体外培养、扩增出神经干细胞。5%的胎牛血清可以诱导神经干细胞向神经元、星形胶质细胞和少突胶质细胞等成熟细胞分化。

Objective To establish methods of cultivation, amplification, differentiation and identification in fetal rats＇ cortical neural stem cells （NSC）. Method 14d fetal rats＇ cerebral cortex was selected as source of cells. To establish the vitro system of embryo neural stem cells,the B27 , basic fibroblast growth factor and vascular endothelial growth factor were added in serum of free medium, 5% of the womb bovine serum was used for differentiation. Immune fluorescence staining techniques were used in identification of neural stem cells and differentiated cells. Results The isolated cultivation of neural stem cells had strong refraction, the cultured cells began to form colonies from 2nd to 3rd which form large ueurospheres. Cells of 3 to 5 passages grew stable. The neurospheres which inducted by fetal bovine serum, from 3rd, began to give prominence to the surrounding and suspension cells begin to grow adherent. The neurospheres of original and passage generation expressed Nestin positive, cells of differentiation expressed NSE, GFAP respectively and expressed GALC positive. Conclusion By using serum-free culture technology we can culture, amplify neural stem cells in vitro. 5% of the womb bovine serum can induce neural stem cells differentiate to neurons cells, astrocytes cells and oligodendroeytes cells and other mature cells.