SD乳鼠原代皮质神经元及星形胶质细胞同时培养的实验方法  

Experimental method of simultaneous culture of primary cortical neurons and astrocytes of Sprague-Dawley suckling rats

作　　者：明江 廖益东 宋文学 穆德勇 刘宗伟 徐卡娅 Ming Jiang;Liao Yidong;Song Wenxue;Mu Deyong;Liu Zongwei;Xu Kaya(Guizhou Medical University,Guiyang 550025,Guizhou Province,China;Department of Neurosurgery,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学,贵州省贵安新区550025 [2]贵州医科大学附属医院神经外科,贵州省贵阳市550004

出　　处：《中国组织工程研究》2023年第15期2339-2343,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金项目(81901173,82060231),项目负责人:徐卡娅;贵州省普通高等学校青年科技人才成长项目(黔教合KY字[2021]190),项目负责人:徐卡娅;贵州医科大学2018年度学术新苗培养及创新探索专项项目(黔科合平台人才[2018]5779-44),项目负责人:徐卡娅。

摘　　要：背景:目前获取原代皮质神经元和星形胶质细胞的方法很多,传统方法通常是分别获取这两种细胞,但实验方法过于繁琐且浪费实验材料。找到一种简单、经济、可行并同时提取这两种细胞的培养方法尤为重要。目的:观察同时提取培养SD乳鼠大脑皮质神经元及星形胶质细胞的效果及注意事项。方法:选用出生24 h内的SD乳鼠,用体积分数为75%乙醇浸泡消毒,待乳鼠昏迷后断脊处死,沿乳鼠颈部用剪刀离断头颅并放入装有高糖DMEM的小烧杯中,沿矢状缝打开头颅并取出大脑放入盛有高糖DMEM的培养皿中。在冰上剥出脑膜及血管,夹取皮质表面2.0-3.0 mm的脑组织,通过木瓜酶和DNA酶进行消化20 min,用含有体积分数为10%胎牛血清的DMEM/F12终止消化。以细胞浓度为1.0×10^(9) L^(-1)种植于含有爬片(用多聚赖氨酸处理)的6孔板中,分别于1,3,5,7 d通过倒置荧光显微镜下观察神经元的形态变化,使用Calcien-AM进行活细胞染色观察细胞活性,β-Tubulin、MAP2免疫荧光染色对神经元进行鉴定。将剩余大脑皮质进行星形胶质细胞的提取,经过胰酶消化、过滤、吹打,以适宜的浓度接种于培养瓶中,待细胞融合度达到90%时,进行恒温水平摇床振荡24 h以纯化星形胶质细胞。当细胞传代纯化后以1.0×10^(9) L^(-1)接种于6孔板中培养,GFAP免疫荧光染色对星形胶质细胞进行鉴定。结果与结论:①培养1 d后可见神经元贴壁生长,胞体增大,部分聚集生长,细胞间有少量的突触连接;培养3,5 d后胞体进一步增大,突触增粗伸长相互连接;培养7 d后神经元明显聚集,突触增长增粗,相互连接形成致密的细胞网。Calcien-AM活细胞染色可见神经元活性较好,经β-Tubulin、MAP2免疫荧光鉴定细胞纯度大于90%。②培养3 d后星形胶质细胞胞体增大,呈现梭状、不规则状,细胞间出现少量相互连接,细胞间存在较多小胶质细胞;培养5 d后胞体、突起�BACKGROUND:At present,there are many methods to obtain primary cortical neurons and astrocytes.The traditional method is usually to obtain these two kinds of cells separately,but the experimental method is too cumbersome and wastes experimental materials.It is particularly important to find a simple,economical,and feasible culture method that simultaneously extracts both types of cells.OBJECTIVE:To observe the effect and precautions of simultaneously extracting neurons and astrocytes from the cerebral cortex of Sprague-Dawley suckling rats.METHODS:The suckling rats within 24 hours of birth were selected and sterilized with 75%alcohol.After the suckling rats were in a coma,they were sacrificed by cutting their spines.The head was severed with scissors along the neck of the suckling rats and placed in a small beaker containing high-glucose DMEM.The skull was opened through the suture and the brain was removed and placed in a petri dish containing high glucose DMEM.The meninges and blood vessels were peeled off on ice,and the brain tissue 2.0-3.0 mm on the surface of the cortex was clipped,digested with papain and DNase,and terminated with DMED/F12 containing 10%fetal bovine serum.Samples were seeded at a density of 1.0×10^(9) /L in six-well plates containing slides(treated with polylysine).The morphological changes of neurons were observed under an inverted fluorescence microscope at 1,3,5,and 7 days,respectively.Calcien-AM was used for live cell staining to observe cell viability,andβ-Tubulin and MAP2 immunofluorescence staining was used to identify neurons.Astrocytes were extracted from the remaining cerebral cortex.After trypsin digestion,filtration,and pipetting,they were inoculated into culture flasks at a suitable concentration.When the cell confluency reached 90%,the astrocytes were shaken on a constant temperature horizontal shaker for 24 hours to purify the astrocytes.After the cells were passaged and purified,they were seeded in six-well plates at 1.0×10^(9) /L.Astrocytes were identified by GFAP immuno

关 键 词：皮质神经元 星形胶质细胞 DNA酶 免疫荧光 原代培养 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R651