小鼠中脑神经干细胞体外培养方法的研究.docVIP

小鼠中脑神经干细胞体外培养方法的研究 肖莉，石清明2，毕文杰1， （1?成都医学院人体解剖学教研室四川省成都市，610083； 2 成都军区疾病预防控制中心,四川省成都市，610021） 【摘要】 ：改进小鼠中脑神经干细胞分离培养方法。：分别采用机械法和酶消化法分离出生1-3天的小鼠中脑组织，在含体积分数为2% B27及2 0ng/ml bFGF的DMEM/F12培养基中进行原代和传代培养。采用巢蛋白（Nestin）、胶质纤维酸性蛋白（GFAP）、及神经元特异性烯醇化酶（NSE）免疫细胞化学染色方法鉴定神经干细胞和分化细胞。：在含bFGF及B27的DMEM/F12培养基中，可形成悬浮生长Nestin呈阳性的神经球。诱导分化的细胞可见GFAP或NSE免疫反应阳性细胞。机械法较酶消化法可获得较多的神经干细胞。：在DMEM/F12培养基中添加2％ B27及20 ng/ml的bFGF可成功获得中脑神经干细胞，机械法为一种简便而有效的神经干细胞分离方法。 【关键词】 小鼠；中脑神经干细胞；分离；培养 Research of?the Mouse?Mesencephalic?Neural?Stem?Cells Cultured in Vitro XIAO Li1，SHI Qingming2，BI Wenjie1，SU?Bingyin1* （1．Department?of?Anatomy? of?Sichuan?Province,?Chengdu?Medical? College，Chengdu，Sichuan，610083，China；2．Center?for?Diseases?control?of?Chengdu?Military?Area，Chengdu，Sichuan，610021，China） 【Abstract】 Objective：The aims of this thesis is to improve the separation and cultivation methods of the mouse mesencephalic neural stem cells．Methods：Using mechanical method and enzyme digestion methods，the mouse mesencephalic tissues are separated after birth from 1 to 3days．After being cultured or subcultured in DMEM/F12 culture medium which have a volume fraction of 2% B27 and 20ng/ml bFGF，immunocytochemical staining method such as Nestin，glial fibrillary acidic protein（GFAP），and neuron specific enolase（NSE）are used to identify neural stem cells and differentiated cells．Results：In the DMEM / F12 culture medium which contain bFGF and B27，Nestin positive neurosphere is suspension growing．The GFAP or NSE immunoreactivity positive cells can be found in the cells after induction of differentiation．Compared with enzyme digestion method，the mechanical method can obtain more neural stem cells. ConclusionAdding 2% B27 and 20ng/ml bFGF into DMEM / F12 culture medium can obtain mesencephalic neural stem cells successfully．The mechanical method is a more simple and more effective method in neural stem cells separation。 【Key words】mouse；mesencephalic neural stem cells；separate；culture （neural stem cellsNSCs）分别采用机械法和酶消化法1～3 1 材料和方法 1