小鼠胚胎神经干细胞的体外培养与鉴定

Culture and identification of neural stem cells from mouse embryos

目的通过改进和优化神经干细胞(NSCs)的体外培养方法,为进一步研究NSCs的生物学行为提供基础。方法孕14 d的美国癌症研究所(ICR)小鼠,无菌条件下剪取前脑皮质,采用机械法分离原代细胞进行培养,采用机械法复合酶消化法对原代细胞进行传代和培养。以1%胎牛血清诱导分化。应用免疫荧光方法行巢蛋白(nestin),β-微管蛋白Ⅲ(β-tubulinⅢ),胶质纤维酸性蛋白(GFAP)染色,对培养的细胞进行鉴定。结果从胚胎小鼠前脑皮质中分离得到的细胞具有连续传代形成克隆球的能力,克隆球呈nestin免疫反应阳性;加入胎牛血清可诱导分化为β-tubulinⅢ和GFAP阳性细胞。结论机械法分离胚胎小鼠前脑皮质细胞,机械法复合酶消化法对原代细胞进行传代和培养,能够成功获得胚胎小鼠前脑的NSCs。

Objective The purpose of this study was to culture and identify neural stem cells from mouse embryos in vitro using a modified method and provide a basis for further study of the biology of neural stem cells under hypoxia. Methods The cells were isolated mechanically from the front cortex of fetal Institute of Cancer Research（ICR） mice on embryonic day 14.They were passaged by mechanical dissociation and enzymatic digestion.The neurospheres were identified by immunofluorescent staining of nestin.Cell differentiation was induced by 1% fetal bovine serum and then the cells were identified by immunohistochemistry of β-tubulin III and GFAP. Results The cells obtained from the front cortex of fetal ICR mice had the capacity of forming neurospheres which showed nestin immunoreactive positivity.After being induced by 1% fetal bovine serum,the cells were differentiated into β-tubulin III-positive cells and GFAP-positive cells. Conclusions Using mechanical dissociation of primary cells and mechanical dissociation with enzymatic digestion of primary cells,the NSCs from the front cortex of mouse embryos can be obtained.