神经干细胞体外培养鉴定及诱导分化的表型特征

Neural stem cells:in vitro culture,identification and differentiated phenotype

背景:神经干细胞促进受损中枢神经系统结构和功能再修复具有广阔的应用前景,而进行神经干细胞体外培养鉴定及诱导分化表型的研究是实现这一应用的基础。目的:观察神经干细胞在体外培养条件下的生物学特性和分化表型特点。方法:从新生小鼠海马、嗅球提取神经干细胞。选取3代后稳定的神经干细胞采用Brd U进行标记,并进行Brd U+巢蛋白+Hochest33258免疫荧光复合染色对神经干细胞进行鉴定。体外诱导促使神经干细胞贴壁分化,对分化产生的子代细胞进行Brd U、β-微管蛋白Ⅲ、胶质纤维酸性蛋白、Hochest33258复合免疫荧光染色确定分化表型。结果与结论:来源于新生鼠海马及嗅球的细胞连续传代培养后可形成稳定悬浮的类球状细胞团,且Brd U+巢蛋白免疫荧光双染阳性。神经干细胞体外诱导贴壁分化后可产生β-微管蛋白Ⅲ、胶质纤维酸性蛋白阳性的子代细胞。以上结果表明体外培养的神经干细胞具有很强的自我增殖更新的能力,在培养过程中趋向于形成稳定的神经球,经体外诱导通过不对称细胞增殖、分化产生神经元和星形胶质细胞等细胞表型。

BACKGROUND： Neural stem cells can repair the damaged central nervous system structure and function, which have broad application prospects. It can be realized by studies on in vitro culture, identification and differentiation phenotype of neural stem calls. OBJECTIVE： To observe the biological characteristics and differentiated phenotypes of neural stem cells under induction in vitro. METHODS： Neural stem calls were extracted from the hippocampus and olfactory bulb of newborn mice. After three generations, neural stem cells were labeled with 5-bremo-2-deoxyuridine （BrdU） and identified by immunofluorescence staining of BrdU, nestin and Hochest33258. The differentiation of neural stem cells was induced in vitro and identified by immunofluorescent staining of BrdU, β-tubulinⅢ, glial fibrillary acidic protein and Hochest33258. RESULTS AND CONCLUSION： After passage, neural stem cells from the hippocampus and olfactory bulb of newborn mice could be aggregated into neurospheres that were positive for nestin and BrdU. Under induced differentiation in vitro, neural stem calls gradually turned into daughter cells which positively expressed β-tubulin III or glial fibrillary acidic protein. These findings suggest that neural stem cells have strong self-renewal capacity and have the tendency to form neurospheres during culture in vitro; under in vitro induction, they can differentiateinto neurons and astrocytes through asymmetric cell proliferation and differentiation.