摘要
为探讨简便、高效的大脑皮质星形胶质细胞体外培养方法,本研究取新生24 h内的ICR小鼠大脑皮层,采用物理方法将其分成约1 mm^3,震荡过滤后进行培养。通过拍照的方式记录原代培养1 d、3 d、7 d、14 d、21 d、28 d、35 d和原代培养14 d后再传代培养14 d(记为P2-14 d)细胞形态;通过实时定量PCR和Western blotting比较原代培养1周、2周、3周、4周、5周和原代培养2周后再传代培养2周(即P2-2)的星形胶质细胞内胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)基因和蛋白水平变化。选取GFAP、S100-β和谷氨酸转运蛋白(excitatory amino acid transporter 1,EAAT1)标记星形胶质细胞,微管相关蛋白(microtubuleassociated protein 2,MAP-2)、离子钙接头蛋白-1(ionized calcium-binding adapter molecule 1,Iba-1)和髓鞘相关糖蛋白(myelin associated glycoprotein,MAG)抗体分别标记神经元、小胶质细胞和少突胶质细胞。通过免疫荧光染色鉴定细胞种类及纯度。研究结果显示细胞生长良好,原代培养4周星形胶质细胞内GFAP比2周、3周、5周和传代培养2周的细胞更加稳定。经免疫荧光鉴定,星形胶质细胞纯度在95%以上。本实验采用相对较简单经济的方法培养出高纯度且生理状态相对较稳定的原代星形胶质细胞,该细胞模型不仅可以用于星形胶质细胞生理功能研究,还可以用于中枢神经系统相关疾病的体外研究。
To explore an effective method to culture and identify the astrocytes from mouse cerebral cortex.Primary cultures of astrocytes were prepared from cerebral cortices of newborn ICR mice.The tissue was cut into about 1 mm^3 pieces by physical methods,then vortexed,filtered and cultured.The morphological changes of the cells at differential stages were observed under phase-contrast microscope.The glial fibrillary acidic protein(GFAP)changes in astrocytes cultured at different stages were compared by real-time quantitative PCR and Western blotting.The types and purity of the astrocytes were identified with immunostaining of astrocyte marker(GFAP,S100-βand EAAT1),neuron marker(microtubule-associated protein 2,MAP-2),microglia marker(ionized calcium-binding adapter molecule 1,Iba-1)and oligodendrocyte marker(myelin associated glycoprotein,MAG).The morphology of these cells has the typical characteristics of astrocytes.The GFAP in primary cultured 4 weeks astrocytes is more stable than that of cultured 2 weeks,3 weeks,5 weeks and 2 weeks of passage culture.The purity of astrocytes of mouse cerebral cortex exceeded 95%.The method to culture highly purified and relatively stable physiological state astrocytes was established successfully,which would be a valuable tool for studying the roles of astrocytes in physiological and pathological of central nervous system diseases in vitro.
作者
许寒晓
蒋烨
罗道朋
李强明
令狐艳
李丽娜
于常海
余资江
戈果
Xu Hanxiao;Jiang Ye;Luo Daopeng;Li Qiangming;Linghu Yan;Li Lina;Yu Changhai;YuZijiang;Ge Guo(Department of Human Anatomy,Guizhou Medical University,Guiyang,550025;Neuroscience Research Institute,Peking University,Beijing,100191)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2019年第7期2966-2971,共6页
Genomics and Applied Biology
基金
贵州省科学技术基金项目(黔科合J字[2015]2012号)
贵州省科技计划课题任务书(黔科合SY字[2014]3024号)
贵州省科技合作计划项目(黔科合LH字[2015]7348)
国家自然科学基金(No.81671218)共同资助