小鼠小脑颗粒神经元的原代培养与鉴定  

Primary Cell Culture and Identification of Cerebellar Granule Neurons in Mouse

作　　者：罗道朋 许寒晓 李强明[1] 余资江[1] 贾熙华 李丽娜[2,3,4] 于常海 戈果[1,2] Luo Daopeng;Xu Hanxiao;Li Qiangming;Yu Zijiang;Jia Xihua;Li Lina;Yu Changhai;Ge Guo(Department of Human Anatomy, Guizhou Medical University, Guiyang, Guizhou;Lab for Functional Study of Astocyte, Neuroscience Research Institute and Health Science Center, Peking University, Beijing, 100191;Neuroscience Research Institute, Peking University, Department of Neurobi- ology, School of Basic Medicine, Peking University Health Science Center, Key Discipline for Neuroscience of the Ministry of Education, Key Laboratory for Neuroscience of the Ministry of Education and the Ministry of Public Health, Beijing, 100191;Hal Kang Lif, Corporation Limited., Beijing, 100191)

机构地区：[1]贵州医科大学人体解剖学教研室,贵阳550025 [2]北京大学神经科学研究所、星形胶质细胞功能研究实验室,北京100191 [3]北京大学神经科学研究所、北京大学基础医学院神经生物学系、教育部神经科学重点实验室、卫生部神经科学重点实验室,北京100191 [4]海康生物科技(北京)有限公司,北京100191

出　　处：《基因组学与应用生物学》2018年第6期2615-2620,共6页Genomics and Applied Biology

基　　金：贵州省科技厅社会发展攻关项目(黔科合SY字(2014)3024号);贵阳市科技计划项目(筑科合同(20141001)21号);贵州省科学技术基金(黔科合J字(2015)2012号);贵州省科技合作计划项目(黔科合LH字(2015)7348号)基金共同资助

摘　　要：本研究通过体外分离培养及鉴定原代小鼠小脑颗粒神经元（cerebellar granule neurons,CGNs）,为神经科学研究提供原代神经元细胞模型。取生后第7天的ICR乳鼠小脑,在解剖体视镜下剥离脑膜及血管,经刀片切碎、0.25%胰酶消化、移液器吹打、70μm尼龙滤网制备单细胞悬液,差速贴壁后接种于新鲜配置的培养基。倒置相差显微镜观察不同时间CGNs的形态变化。采用神经元的标记蛋白微管相关蛋白-2（microtubule associated protein 2,MAP2）,星形胶质细胞的标记蛋白胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）,小胶质细胞的标记蛋白（type 3 complement receptor,CR3/CD11b）进行免疫荧光检测培养的原代CGNs纯度。原代培养CGNs接种20 min后即贴壁良好,培养24 h后细胞伸出突起,培养第7天神经元成熟,形成丰富的轴突,树突和胞间突触连接。经免疫荧光鉴定,CGNs纯度可达95%以上。成功体外分离培养出高纯度的原代CGNs,可应用于CGNs的体外研究。Isolation and identification of primary culture of cerebellar granule neurons（CGNs） in vitro, for neuroscience experiments to provide primary neurons model. Cerebellum was removed from a postnatal 7 days old ICR mouse, meninges and blood vessels were cleaned carefully under stereoscopic microscope. The cerebella were minced with surgical blade, digested with 0.25% trypsin, dissociated by pipetting and filtered with 70 μm nylon mesh to obtain single cell suspension. Granule cells were purified by differential velocity adherence method and cultured in fresh medium. The morphological change of cultures at differential stages was observed under phasecontrast microscope. Characterization and purity of the neurons were determined by immunostaining of neuron marker（microtubule associated protein 2, MAP2）, astrocyte marker（glial fibrillary acidic protein, GFAP） and microglia marker（type 3 complement receptor, CR3/CD11b）. Isolated CGNs adhered well to the substrate after 20 mins,cells started to extend axons and dendrites after 24 hours. The neurons matured and form neural network at day 7.The purity of cultured CGNs was determined to be more than 95 percent by using immunofluorescent technique.The high purity primary culture of CGNs had been successfully obtained and could be used for in vitro studies.

关 键 词：小脑颗粒神经元 小鼠 原代培养 免疫荧光 高纯度 

分 类 号：R338[医药卫生—人体生理学]