神经干细胞的分离培养与鉴定

目的　分离培养新生鼠神经干细胞并获得细胞克隆 ,同时建立良好的传代方法。方法　取新生大鼠大脑室管膜、脑室周围及海马细胞 ,经消化及机械吹打后接种于加有N2添加剂及生长因子的DMEM/F12 培养基 ,悬浮培养 ;用含血清而不含生长因子的培养基贴壁培养 ,促使分化 ;最后用免疫细胞化学方法进行染色鉴定。结果　分离培养的细胞多聚集生长 ,并形成细胞克隆 ,可以传代 ;细胞为圆形或椭圆形 ,经鉴定为nestin抗原阳性 ;分化后细胞分别表达神经元、星形胶质细胞和少突胶质细胞特异性抗原。结论　分离培养的细胞具有自我更新和多向分化能力 ,表达神经干细胞的特异性抗原nestin ,是神经系统的干细胞。

Isolation, culture and identification of neural stem cells in rats

Objective To isolate and culture neural stem cells from SD newborn rats and to establish a satisfactory method of passage. Methods We isolated the cells from the ependyma of cerebral ventrical, subventricular zone and hippocampus and then planted them into DMEM/F 12 culture medium with N2 and growth factors to suspension culture after digestion or mechanical dissociation. These cells were planted into DMEM/F 12 culture medium with serum (without growth factors), then were passaged and induced to differentiate. The stem cells, neurons, astrocytes and oligodendrocytes were evaluated with immunocytochemistry by nestin, β-tubilin, GFAP and GalC, respectively. Results The cells in this study grew together and formed neurospheres. They were round or oval and could be passaged. These cells were nestin positive cells. The differentiated cells were neurons, astrocytes and oligodendrocytes. Conclusion The cells we cultured are nestin positive cells and have the capacity of self-renewal, can give rise to neurons, astrocytes and oligodendrocytes. It is suggested that they are neural stem cells.