神经干细胞的分离培养和鉴定及体外长期培养

Isolation,identification and long-term ex vivo cultivation of the neural stem cells

目的探索新生SD大鼠神经干细胞的分离培养、鉴定及体外长期培养的方法。方法分离新生SD大鼠海马组织,用无血清培养技术培养神经干细胞,免疫组织化学荧光技术检测其巢蛋白(Nestin)的表达;并用5-溴脱氧尿嘧啶核苷(BrdU)掺入试验,免疫荧光双标技术观测神经干细胞的增殖状况。诱导神经干细胞分化,分别用神经元特异性核抗原(NeuN)抗体、胶质纤维酸性蛋白(GFAP)抗体、2,3-环核苷酸磷酸二脂酶(CNPase)抗体进行免疫组织化学荧光染色鉴定分化细胞。结果获得大量未分化呈巢状悬浮生长的神经干细胞团,并能分化为神经元和神经胶质细胞,且经传代培养10代以上仍具干细胞特性。结论成功培养出胚胎大鼠神经干细胞,培养出的细胞具有增殖、自我更新及多潜能分化能力,可分化为神经元及神经胶质细胞并可作为神经干细胞移植实验研究的供体细胞。

[ Objective ] To investigate the isolation, identification and long-term ex vivo cultivation of the neural stem cells （NSCs） from neonatal rats. [Methods] Hippocampus was isolated from neonatal SD rats and NSCs were cultured in serum free medium. Nestin expression in NSCs was detected by immunohistochemical fluorescence. The proliferation of NSCs was assessed by both of 5-bromodeoxyuridine （5-BrdU） incorporation assay and double-marked immunofluorescence. The differentiation of NSCs was induced. To identify the dif- ferentiated NSCs, antibodies of neuron specific neuronal nuclei （NeuN）, 2, 3-cyclic-nucleotide Phosphodi- esterases （CNPase） and antibody of glial fibrillary acidic protein （GFAP） were employed respectively with im- munohistochemical fluorescence staining. [Results] A mass of undifferentiated neurospheres were obtained and cultured in suspension like nests. Those NSCs could differentiate into neurons and astrocytes, and still possess the properties of stem cells after more than 10 passages. [Conclusion] We successfully obtained the fetal rat NSCs, which have the capacities of proliferation, self-renew and pluripotentiality. They could differentiate into neurons and astrocytes. They are the potential donor cells for neural stem cells transplantation.