胎鼠脊髓神经干细胞的分离培养、鉴定与诱导分化

Isolation,culture and identification of spinal cord-derived neural stem cells

目的探讨胚胎大鼠脊髓神经干细胞分离、培养、传代及鉴定的方法。方法显微解剖分离E14d胚胎大鼠脊髓组织,用机械吹打法制成单细胞悬液,接种于含有表皮生长因子(EGF)、碱性成纤维生长因子(bF-GF)及N2的DMEM/F12(1∶1)无血清培养基中培养,待形成克隆球后进行传代培养。取第3代培养的细胞分别进行Nestin免疫细胞化学鉴定和诱导分化后β-Ⅲ-tubulin、GFAP、CNPase免疫细胞化学鉴定。结果E14d胚胎大鼠脊髓组织细胞培养72h后可形成悬浮生长的神经球,7～10d后即可传代。经传代扩增后得到大量同源的Nestin免疫阳性的细胞克隆球,经诱导分化后大部分细胞分化为GFAP免疫阳性的星形胶质细胞和CNPase免疫阳性的少突胶质细胞,少量细胞分化为β-Ⅲ-tubulin免疫阳性的神经元。结论成功从胚胎大鼠脊髓组织中分离出神经干细胞,为进一步开展脊髓疾病的细胞移植治疗研究奠定了基础。

Objective To culture and identify E14 rattus spinal cord neural stem cells, Methods The spinal cord was dissected from embryonic 14d rats under anatomical microscope. Using a fire-polished glass pipet, the spinal cord tissue was triturated into a fine single-cell suspension. The cells were cultured with the serum-free medium containing DMEM/F12 （ 1 ： 1 ） , N2 supplement, EGF and bFGF. After the primary neurospheres formed, the single-cell suspension from primary neurospheres was obtained by mechanical separation method and then the Cells were subcultured. The neural stem cells of the third passage were identified with immunocytochemistry for Nestin. At the same time, the cells were induced to differentiation. The distinctive marker for neuron （ β-Ⅲ-tubulin）, astrocyte （GFAP） and oligodendrocyte （CNPase） were employed to detect the phenol type of differentiated cells. Results 72h after culturing,the cells derived from E14 rat embryonic spinal cord form neurospheres,which can be subcultured 7 -10 days after culturing. A great many of neurospheres can be ob- tained by successive passage. The immunocytochemistry showed the cells were Nestin positive, after differentiation, the cells expressed GFAP,CNPase and β-Ⅲ-tubulin. Conclusion The neural stem cells derived from E14 rat embryonic spinal cord are successfully isolated and cultured, which are multipotent and have the ability to self-renew and will provide the foundation for the further research about cell therapy of spinal cord＇ s disease.