大鼠海马神经元neurobasal无血清的原代培养方法  

Establishing a serum-free primary culture method for hippocampal neurons of rats

作　　者：王圆圆[1] 张文斌[2] 龚晓兵[2] 郭国庆[1] 陈静[1] 辛莉[1] 沈伟哉[1] 原林[3] 

机构地区：[1]暨南大学医学院解剖学教研室,广东广州510632 [2]暨南大学第一附属医院,广东广州510630 [3]南方医科大学解剖学教研室,广东广州510515

出　　处：《暨南大学学报（自然科学与医学版）》2008年第6期547-551,共5页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家973基础研究计划(2007CB512705);广东省自然科学基金项目(8451063201000193);广东省医学科研基金项目(A2008344)

摘　　要：目的：建立纯度和活力较高的无血清原代培养海马神经元的方法。方法：新生SD大鼠海马，用neurobasal培养基培养，免疫荧光鉴定神经元纯度，MTT法检测其活力。结果：神经元接种12～24h后贴壁，并长出细小突起，3d具有典型神经元形态特征，4d突起形成稀疏的神经纤维网络，8d后神经元5～10个聚集成团，突起密集，生长稳定，12d后出现细胞碎片。Tubulin荧光染色显示清晰的神经元，突起绵长且相互交织，占细胞总数的66．7％；GFAP荧光染色的细胞数量少，突起短粗，占33．7％。培养1～5d MTT代谢率逐渐上升，6～11d处于平台期，11d后下降。结论：neurobasal无血清培养获得的神经元纯度大于60％，6～11d的细胞适于细胞学实验。Aim： To establish a serum-free primary culture method for hippocampal neurons of rats. Methods ： Hippocampal neurons of neonatal SD rats were cultured by neurobasal medium, then the purity and activity of neurons were investigated by immunofluorescence and MTT assays. Results ： Neurons were adhesion and developed small neurites after cultivated for 12 -24 hours, then a typical neuronal morphology appeared after 2 -4 days, up to 8^th day, many neurites extended to form sparse network , meanwhile, cells gathered together and neurites were intensive, at last, cell fragment was obsevered in 4 clays later. Fluorescence staining with anti-tubulin antibody showed clear neurons and abundant long neurites, moreover, the coloring gradually weaker away and the neurons made up 66.7% of total cells; on the contrary, fluorescence was light and neurites were short with GFAP staining, the proportion was 33.7% ; MTT increased in 1 -5 days, stablizing from 6^th day to 11^th day and finally dropping after 11 days. Conclusion： The purification rate of neurons cultured by neurobasal serum-free meduim is more than 60% and cells cultured after 6 - 11 days were suitable to observation.

关 键 词：神经元 原代培养 neurobasal培养基 海马 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] Q954.6[医药卫生—基础医学]