人胚胎神经干细胞体外培养及其增殖与分化的研究

Cultivation,proliferation and differentiation of human embryonic neural stem cells in vitro

目的 建立神经干细胞分离、培养及分化的鉴定技术 ,观察神经干细胞增殖、分化的特点。方法 从人胚胎海马区分离神经干细胞 ,采用无血清培养基 ,进行体外扩增培养、传代。采用免疫细胞化学法鉴定神经干细胞和分化的神经细胞 ;利用流式细胞仪和细胞生长曲线检测神经干细胞的增殖能力。结果 从人胚胎脑海马区分离的细胞具有增殖和多向分化潜能 ,可进行传代培养 ,获得的细胞团中大部分为nestin表达阳性细胞。贴壁分化后可以出现NSE、GFAP表达阳性的细胞。结论 用上述方法分离培养的细胞能表达nestin蛋白 ,具有自我更新和增殖能力 ,并具有向神经元、星形胶质细胞分化的潜能 ,具备神经干细胞的特征 。

Objective To establish the methods of isolation,cultivation and in vitro differentiation of neural stem cells and to observe the characteristics of proliferation and differentiation of neural stem cells.Methods The serum free culturing technology was used to isolate,culture and pass neural stem cells from embryonic human hippocampus.Immunocytochemistry,flow cytometry and cell growth curve were used to identify the neural stem cells and their potentiality of differentiation and proliferation.Results The neurospheres isolated from embryonic human hippocampus possess the ability to proliferate,self renew and passage.Nestin positive cells can be found in the neurospheres.After attachment they can differentiate into GFAP and NSE positive cells.Conclusion The methods to isolate and culture neural stem cells from embryonic human hippocampus are established.The cultured neurospheres possess the capability of proliferation,self renew and multipotentiality.The neurospheres that express nestin are the neural stem cells.It can be used to study on cell transplantion.