细胞质膜微囊蛋白1敲除小鼠神经干细胞培养与生物学特性  

Culture and biological characteristics of neural stem cells from caveolin-1 knockout embryonic mice

作　　者：刘柏炎[1,2] 俞悦[1] 易健[1] 陈雪梅[1] 蔡光先[1] 

机构地区：[1]湖南中医药大学,湖南省长沙市410007 [2]益阳医学高等专科学校,湖南省益阳市413000

出　　处：《中国组织工程研究》2014年第23期3739-3744,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(30873355;81273989);国家重点基础研究发展计划(973计划)(2010CB530400);2012年度湖南省高校创新平台开放基金项目(12K089)~~

摘　　要：背景:研究发现细胞质膜微囊蛋白1在哺乳动物脑内表达,参与脑的正常发育,能影响脑内神经干细胞的增殖。目的:从细胞质膜微囊蛋白1敲除小鼠脑内获取神经干细胞并观察其生物学特性。方法:分别取E14-E16正常C57BL/6胚胎小鼠和细胞质膜微囊蛋白1敲除C57BL/6胚胎小鼠全脑,采用酶消化法获得单细胞悬液,置神经干细胞条件培养基中培养,原代培养7 d后,加入含体积分数10%胎牛血清的DMEM/F12培养基诱导7 d。结果与结论:从两种胎鼠脑内获取的细胞悬液培养1 d后较多细胞发生死亡,可见单个细胞漂浮于培基中,透光度较好,3 d后逐渐形成悬浮生长的多细胞团。传代后培养板底部可见少量细胞发生贴壁,7 d后可见大量细胞团出现,且细胞质膜微囊蛋白1敲除胎鼠源细胞增殖速度更明显。免疫细胞化学检测显示,两种胎鼠来源细胞团均为巢蛋白阳性,分化细胞可见神经丝蛋白200、胶质纤维酸性蛋白或O4阳性表达;正常C57BL/6胎鼠来源细胞团呈细胞质膜微囊蛋白1阳性表达,而细胞质膜微囊蛋白1敲除C57BL/6胎鼠来源细胞团呈细胞质膜微囊蛋白1表达阴性,且神经干细胞成球速度和成球数量优于正常胎鼠神经干细胞。说明实验成功从细胞质膜微囊蛋白1敲除胎鼠脑内培养出细胞质膜微囊蛋白1缺失神经干细胞,细胞质膜微囊蛋白1可促进神经干细胞的增殖,并抑制其分化。BACKGROUND：Caveolin-1 is expressed in mammalian brain and involved in the normal development of the brain, which can affect the proliferation of neural stem cells in the brain. OBJECTIVE：To acquire neural stem cells from caveolin-1 knockout embryonic mice in vitro and study their biological characteristics. METHODS：The whole brain was separated from C57BL/6 mice and caveolin-1 knockout C57BL/6 mice respectively at encyesis 14-16 days. Single cellsuspension was obtained by enzyme digestion, and cultured in the conditioned medium of neural stem cells. Fol owing 7 days of primary culture, the cells were induced in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10%fetal bovine serum for 7 days. RESULTS AND CONCLUSION：The major cells of the cellsuspensions from the fetal mouse brain were dead at 1 day after culture, and some single cells floated in the medium and their transmittance were better, and then they gradual y formed multicellular bal s after 3 days. A smal amount of cells were adhered at the bottom of culture plate after passage, and a great amount of cellbal s appeared after 7 days. The proliferation rate of neural stem cells from caveolin-1 knockout mice was higher than that from normal mice. The cellbal s were nestin-positive and their differentiated cells was positive for neurofilament 200, glial fibril ary acidic protein or O4, respectively. Al of the cells from normal mouse brain were positive for caveolin-1, but the cells from caveolin-1 knockout mice were negative for caveolin-1 by immunocytochemistry. Moreover, the speed of cellbal formation and the number of cellbal s in neural stem cells from caveolin-1 knockout mice were better than those from normal mice. Caveolin-1 negative neural stem cells were cultured successful y from caveolin-1 knockout mouse brain, and the results show that caveolin-1 can promote the proliferation of neural stem cells and inhibit their differentiation in vitro.

关 键 词：干细胞 培养 神经干细胞 细胞质膜微囊蛋白1 生物学特性 C57BL 6胎鼠 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]