胚胎小鼠神经干细胞的培养和诱导分化  

The Culture and Induced Differentiation of Neural Stem Cells of Embryonic Mice

作　　者：任丽伊 赵海霞 左璇[1] 李淑蓉 苏炳银 

机构地区：[1]成都医学院基础医学院,成都610500 [2]发育与再生四川省重点实验室,成都610500

出　　处：《成都医学院学报》2017年第1期1-6,共6页Journal of Chengdu Medical College

基　　金：国家自然科学基金资助项目(No:31600973);发育与再生四川省重点实验室基金资助项目(No:SYS14-003);四川省教育厅科研基金资助项目(No:16ZA0278);成都医学院校基金资助项目(No:CYZ14-004)

摘　　要：目的探索体外培养和诱导分化胚胎小鼠神经干细胞(neural stem cells,NSCs)的方法。方法分离12.5dICR小鼠胚胎的大脑皮质,通过Accutase酶消化法和机械消化法,将组织消化成单个细胞,置于完全培养基中培养,3d后神经干细胞增殖成神经球;将传代的神经球消化,通过诱导培养基诱导其分化;采用细胞免疫荧光染色和qRT-PCR技术检测巢蛋白(Nestin)、中间微管相关蛋白2(Map2)和胶质纤维酸性蛋白(Gfap)的表达,鉴定NSCs、分化形成的神经元和星形胶质细胞。结果 NSCs能被Nestin特异性标记,诱导分化后的细胞可被Map2和Gfap抗体特异识别;qRT-PCR结果显示,诱导后Nestin的表达水平降低,而Map2和Gfap的表达水平升高。结论通过Accutase酶消化法联合机械消化法分离小鼠胚胎脑皮质,进行体外培养,可获得具有自我更新能力和多向分化潜能的NSCs。Objective To explore the method of culture and induced differentiation of neural stem cells in vitro.Methods The neural stem cells were isolated from the cerebral cortex of 12.5-day mouse embryo and digested into single cells by the Accutase enzyme digestion method and mechanical percussion.The single cells were cultured in complete medium.After 3 days,the neural stem cells proliferated into neurospheres.The subcultured neurospheres were digested and differentiated by induced medium.The methods of immunofluorescence staining and qRT-PCR were adopted to assay the expression levels of Nestin,microtubule-associated protein 2 （Map2）,and glial fibrillary acidic protein （Gfap）and identify the neural stem cells and the neurons and astrocytes differentiated from the neural stem cells.Results The cultured neural stem cells were marked by Nestin specific markers;the cells induced from the neural stem cells could be identified by anti-Map2 or anti-Gfap specific antibody.According to the results of qRT-PCR,the expression level of Nestin decreased,while the expression levels of Map2 and Gfap increased.Conclusion The Accutase enzyme digestion method combined with the mechanical percussion can separate the cells from the cerebral cortex of embryonic mouse and those cells can be cultured in vitro to obtain the neural stem cells with the capability of self-renewal and multi-directional differentiation potential.

关 键 词：神经干细胞 神经元 星形胶质细胞 诱导分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]