新生大鼠海马神经干细胞的分离培养、分化与鉴定

Isolation, culture, differentiation and identification of hippocampus-derived neural stem cells in neonatal rats

目的建立新生大鼠海马神经干细胞（NSC）分离培养与鉴定方法。方法分离新生24h内SD大鼠海马组织，制成单细胞悬液，采用含表皮生长因子（EGF）和碱性成纤维细胞生长因子（bFGF）的无血清条件进行培养，1周后传代。结果培养48～72h，细胞聚集悬浮生长，形成典型的神经球，呈神经干细胞巢蛋白（Nestin）阳性表达，并能在体外传代连续形成克隆，原代和传代的NSC加入含10％胎牛血清的DMEM／F12培养基，1～2d后神经球即开始贴壁分化生长，第7天分别呈神经元特异性烯醇化酶（NSE）阳性和胶质纤维酸性蛋白（GFAP）阳性表达。结论新生大鼠海马中存在NSC，分离培养时具有自我更新增殖和多分化潜能。

Objective To establish the methods for culture and identification of neural stem cells i- solated from the hippoeampus of neonatal rats. Methods The hippocampal tissues of 24-h newborn SD rats were separated to produce single cell suspension, which was cultured with serum-free medium supplemented with epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF）, and passaged one week lat- er. Results After primary culture for 48-72 h, cells gathered with suspension growth, and typical neuro- sphere formed, expressed nestin and cOuld be passaged in vitro. Primary and passage cultured NSCs were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum, 1-2 days later neurospheres ad- hered to the wall and differentiated, and at day 7, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） were expressed. Conclusion The NSCs exist in the hippoeampus of newborn rats, and they have potentials of self-renewal and muhipotential differentiation during isolation and culture.