新生大鼠海马神经干细胞的体外培养及鉴定

Culture and identification of neonate rat hippocampus neural stem cells in vitro

目的探讨新生大鼠海马神经干细胞(NSC)的体外培养和诱导分化的条件和特点。方法分离出生1d大鼠海马,在表皮生长因子、碱性成纤维生成因子和B27联合作用下使其稳定增殖,用5-溴脱氧尿苷(BrdU)标记处于增殖状态的神经干细胞,应用免疫荧光染色方法行巢蛋白(Nestin)、5-溴脱氧尿苷(BrdU)、β-Ⅲ型微管蛋白(Tuj-1)、波形蛋白(Vimentin)和Galc-C免疫荧光染色,对NSC的增殖及其分化的细胞进行鉴定。结果体外培养的NSC增殖成神经干细胞球并传代,鉴定为Nestin染色阳性细胞和5-溴脱氧尿苷(BrdU)标记染色阳性细胞,并可诱导分化为神经元细胞(Tuj-1染色阳性细胞)、神经胶质细胞(Vimentin染色阳性细胞)和少突胶质细胞(Galc-C染色阳性细胞)。结论采用无血清培养基中加入特定生长因子的培养技术,可培养出在体外稳定增殖并有多向分化潜能的新生大鼠海马NSC。

Objective To explore the conditions of culturing neonate rat hippocampus neural stem cells in vitro and their inentification.Methods Hippocampus of neonatal rats（ 1 day）were taken out and mechanicaly dissociated and proliferated steadily under the effect of epidermat growth factor （EGF）, basic fibrobhst grewth factor （ bFGF ） and B27. The rat neural stem eeU proliferation was marked by 5-bromedcoxyuridine （Bn：IU）. The proliferation and differentiation of neural stem cells was identified respectively by inanunofluorescenceincluding Nesfin, 5-bromodeoxyufiding（BrdU）,chss Ⅲ β-tubulin （Tuj-1）,Vimentin and Galc-C.Results The cultured neural stem celh proliferated, and become the sphere. The rat neural stem cells after the culture and passage were proved by povifive Nestin and positive cell by BrdU marking. The differentiated cells in the sphere could express specific antigens of neurons （ positive Tuj- 1 ）, glial cell（positive Vimentin） and oligedendrocyte （ positive Galc-C ）. Conclusion With the help of growth factors and serum-free technlque,the neonate rat hippocampus neural stem cells which have potency of multiple differentiations can suceessfully by cultured in vitro.