人胎脑神经干细胞的体外分离、培养与鉴定

Isolation,cultivation and identification of human fetal neural stem cells in vitro

目的探索人胎脑神经干细胞的体外分离培养条件,从而在体外大量增殖神经干细胞,并观察神经干细胞增殖和分化的特点。方法采用含碱性成纤维细胞生长因子(bFGF)和表皮细胞生长因子(EGF)的无血清培养技术,从14周自愿水囊引产人胎脑海马皮质中分离出神经干细胞,并进行培养、传代、分化观察,应用免疫荧光细胞化学技术对培养的细胞及其分化的细胞进行鉴定。结果从14周人胎脑皮质中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈悬浮状态生长,形成神经球,该细胞具有连续增殖能力,可传代培养,表达神经巢蛋白抗原(Nestin);在含血清培养时诱导神经干细胞分化,分化后的细胞表达神经元细胞和胶质细胞的特异性抗原。结论在含有EGF和bFGF的无血清培养液中,从人胎脑能分离培养出具有自我复制和分化潜能的神经干细胞,并能在体外扩增,可进一步用于基础及临床研究。

Objective To explore the methods of isolation,cultivation and differentiation of human fetal neural stem cells,and to observe the characteristics of proliferation and differentiation of neural stem cells in vitro.Methods The neural stem cells were isolated from human fetal hippocampus at embryonic age 14 weeks by induction of labor with water bag.A serum free medium containing basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF） was used to cultured and expanded neural stem cells.The cells were cultured,passed and differentiated.Cultured and differentiated cells were identified with immunofluorescence test.Results The human neural stem cells having the abilities of self-renew and multipotency were successfully isolated and cultured from human fetal brain with serum free medium.They were found to form typical neurospheres in suspension and express Nestin antigen.The cells could be amplified and passed continuously.Neural stem cells were induced to differentiate and express specific antigens of neuron and astrocyte in serum medium.Conclusion The neural stem cells isolated from human fetal brain have the abilities of self-replication,multipotency and amplification in vitro.It has useful appliacation in further basic and clinical research.