大鼠脑皮质星形胶质细胞的体外优化培养及鉴定  

The optimized culture and identification of astrocytes from rat cortical tissue

作　　者：靳辉[1] 冯改丰[1] 杨蓬勃[1,2] 贾宁[1] 杨维娜[1] 钱亦华[1] 王唯析[1] 

机构地区：[1]西安交通大学医学部人体解剖与组织胚胎学系,陕西西安710061 [2]西安交通大学医学部神经生物学研究所,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2015年第6期849-853,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省自然科学基金资助项目(No.2012JQ4011;2014JM4096);中央高校基本科研业务费专项资金资助项目(No.xjj2012011)~~

摘　　要：目的旨在获得高纯度的星形胶质细胞,并对不同培养时期的细胞进行鉴定,为进一步研究奠定基础。方法常规分离新生SD大鼠大脑皮质并制备单细胞悬液,采用差速黏附加摇床震荡法对所获得的细胞进行纯化。倒置相差显微镜及HE染色观察细胞形态,GFAP免疫荧光对获得细胞进行鉴定。结果原代培养的皮质细胞从3d开始迅速增殖,9-12d即可铺满瓶底,此时细胞呈明显的分层生长,星形胶质细胞位于底层,形态多样,阳性率为（67.2±7.1）%;经过1次传代后,GFAP阳性率有所提升（84.0±6.0）%,但不能完全去除小胶质细胞及少突胶质细胞,细胞仍呈分层生长;至第3次传代后,获得大量几乎为单一种类的星形胶质细胞,其胞体较大、突起较短粗,一般2-3个,胞核圆形或椭圆形,常偏于一侧,GFAP染色呈强阳性,阳性率可达（97.6±2.4）%。结论通过差速黏附与摇床震荡相结合的方法,获得了高纯度且生长状态良好的星形胶质细胞。Objective To obtain highly purified astrocytes and identify the cells in each stage to support further studies.Methods The cerebral cortex of a neonatal SD rat was isolated and prepared into single cell suspension.The obtained cells were purified by differential adherence and shook at a constant temperature.By inverted phase contrast microscopy and HE staining,cell morphology was observed.The immunofluorescence staining with anti-mouse GFAP was used to identify the cells.Results The primary cortical cells developed rapidly at 3 d after culture and covered the flasks at 9-12 d.At this time,the cells showed stratification and the astrocytes lay at the lower layer.GFAP positive rate was only about（67.2±7.1）%.After the first passage,GFAP positive rate increased obviously（84.0±6.0）%. However,oligodendrocytes and microglias could not be removed completely,and the cells also showed stratification.Through3 times of passages,we obtained many single species of astrocytes showing satellite shape with2 or 3 processes,big cell body and round or oval-shaped nuclei leaned to one side.Immunofluorescence staining showed that nearly all of the cells were strong positive and the positive rate reached as high as（97.6±2.4）%.Conclusion Through differential adherence and shaking at a constant temperature,more astrocytes of high purity and in good state can be obtained.

关 键 词：星形胶质细胞 大脑皮质 细胞培养 大鼠 

分 类 号：Q25[生物学—细胞生物学]