早期人胚神经干细胞分布及分离培养

A morphological and distributive study of neural stem cells in human embryonic brain and culture of human embyonic neural stem cells in vitro

目的 研究发育早期人胚脑神经干细胞的形态特征及其分布情况 ,并观察其体外生长分化特性。方法 计划生育流产的 6～ 12周胚胎大脑组织切片用vimentin抗体进行免疫组织化学染色 ,光镜观察。机械分离皮质细胞 ,用无血清培养基和含血清培养基进行悬浮和贴壁培养 ,分别用MAP2 、GFAP及GalC抗体进行免疫细胞化学染色 ,鉴定干细胞和神经元、星形胶质细胞及少突胶质细胞。结果 发育早期大脑Vimentin阳性细胞分布广泛 ,细胞形态单一 ,多呈圆球形 ,体积较小。体外培养的大脑皮质细胞在培养的第 5～ 7天时 ,形成较多克隆 ,可多次传代 ;贴壁培养时细胞发生分化 ,分别为MAP2 、GFAP、GalC阳性的神经元、星形胶质细胞及少突胶质细胞。结论 胚胎发育早期大脑神经干细胞分布广泛 ,形态单一 ,皮质Vimentin阳性细胞数量多 ,便于分离 ,是最佳神经干细胞分离部位 ;

Objective To observe the morphology and distribution of neural stem cells(NSC) in first trimester human brain of pregnancy and to study the proliferation and differentiation features of human NSCs in vitro . Methods Six fetal brains of first trimester were fixed with formaldyhydum polymerisatum and embeded with paraffin wax. Then, morphology and distribution of the NSCs were observed after immunohistochemistry stain with vimentin. The cerebral cortex cells were dissociated mechanically and planted into DMEM/F 12 culture medium supplemented with N2 and growth factors to suspension culture. Then these cells were induced to differentiate after culture in DMEM/F 12 medium with FBS. The stem cells, neurons, astrocytes and oligodendrocytes were identified by immunocytochemistry with antibodies vimentin, MAP 2, GFAP and GalC, respectively. Results The vimentin immuno reactive cells distributed extensively in all the regions of fetal brain. They were small, round or ovoid in shape. The cerebral cortex cells were cultured in vitro with growth medium and formed neurospheres at 5th～7th day after being planted. These cells were passaged many times and then formed subclones. After adherent culture in the medium with FBS, the vimentinir cells differentiated spontaneously into neurons (MAP 2 ir), astrocytes (GFAP ir) and oligodendrocytes (GalC ir). Conclusion The NSCs distribute extensively in human embryonic brain. A large number of stem cells exist in the cortex and they can be isolated easily. These cells are expantioned successfully in vitro and they differentiate into three major types of cells of the nervous system.