Study on cell lineage of neuronal stem cell in vitro

碱性成纤维细胞生长因子（bFGF）和表皮生长因子（EGF）刺激神经干细胞的增殖，从早期发育的大脑。对于细胞上的生长因子受体，在培养体系中表达的时机似乎影响其细胞谱系。本研究主要针对神经干细胞的细胞谱系进行研究。从胚胎12天的前脑中分离细胞。建立了流式细胞仪结合光散射技术分离细胞的方法。该方法的分离质量高，可从细胞悬液中纯化神经干细胞，对培养体系中的细胞事件有重要影响。在前脑细胞悬液中，A_2B_5结合细胞占总细胞的12%，而绝大多数细胞为nestin阳性细胞，两种抗体的结合细胞也出现在悬液中。分别在通过流式细胞术分离的细胞中在分化培养基中进行细胞培养。这些细胞群首先生成神经元细胞，然后生成神经胶质细胞。这些细胞随机产生神经祖细胞和神经胶质祖细胞。这一结果表明，胶质祖细胞在培养8 - 10天时不接受FGF信号，而在含EGF的培养基中，神经干细胞在培养8 - 10天时出现神经球，而在培养8天之前则未出现神经球。EGF和FGF对细胞的刺激作用不同。

Basic fibroblast growth factor(bFGF) and epidermal growth factor(EGF) stimulates the proliferation of neural stem cells from early developmental brain. For the receptor of the growth factor on the cells, the timing of expression in culture system is seem to affects its cell lineage. In this study, focus on cell lineage of neural stem cells. The cells are separated from forebrain in embryonic stage at 12 day. The cell isolation method is established to use flow cytometry with light scattering. Isolation qualify in this method would be highly to purify neural stem cells from the cell suspension which effects to cellular events in culture system. In cell suspension of forebrain, A2B5 bound cells were appeared 12% in total cells, whereas almost cells were belonged to nestin-positive cells.The bound cells in both antibody were also appeared in the suspension. Cell culture is performed in differentiation medium in cells isolated by flow cytometry, respectively. These cell populations were generate neuronal cells first and glial cells later. The cells generate neural progenitor and glial progenitor cells randomly. This result would seem that glial progenitor was not accepted FGF signaling in culture period at 8 to 10 days.In medium containing EGF, neural stem cells were appeared neurospheres at 8 to 10 days, whereas not before 8 day. It seem to be a different between FGF and EGF stimulate cells.