新生小鼠神经干细胞体外培养、分化及鉴定

In vitro culture, differentiation, and identification of neural stem cells in neonatal mice

目的简化神经干细胞原代培养的取材及步骤,明确体外定向诱导分化条件,为进一步神经干细胞移植相关实验提供基础条件。方法新生24 h昆明种小鼠无菌条件下冰上取大脑组织,经机械分离加胰酶消化吹打后,加入含有碱性成纤维细胞生长因子、表皮细胞生长因子和B27的DMEM/F12培养基中培养;加入不同浓度胎牛血清诱导其分化,应用免疫荧光技术行巢蛋白、胶质纤维酸性蛋白、微管相关蛋白2染色,对培养细胞鉴定。结果新生小鼠提取神经干细胞可形成神经球,并稳定增殖传代,经诱导可定向分化为神经元及星形胶质细胞。结论新生小鼠较胎鼠取材简单,可培养高质量神经干细胞,血清浓度同向星型胶质细胞方向的分化概率呈正相关。

Objective To simplify the original culture of neural stem cells in vitro and to determine the differentiation conditions. Provide basic conditions for the further study of neural stem cell transplantation. Methods The brain tissue of Kunming mice newborn for 24 h was isolated under sterile conditions on the ice, by mechanical separation and trypsin digestion and trituration, and added with alkaline fibroblast growth factor, epidermal growth factor, and B27 of DMEM/F12 culture medium; The brain tissue was also added with different concentration of fetal bovine serum to induce differentiation; The immune fluorescence technique for nestin, glial fibrillary acidic protein, and microtubule associated protein 2 staining of cell culture identification were also performed. Results The primary cultured cells of the neonatal mice could form the nerve bulb and proliferate and differentiate into neurons and astrocytes. Conclusion Compared with the fetal rats, the newborn mice are simple and neural stem cells with high quality could be cultured, and the serum concentration is positively correlated with the differentiation of the direction of the astrocytes.