胎鼠大脑皮质神经元的分离培养和鉴定  

Isolation and identification of neurons from the cerebral cortex of embryonic rats

作　　者：周竹娟[1] 郑健[1] 

机构地区：[1]第三军医大学新桥医院神经内科,重庆400037

出　　处：《卒中与神经疾病》2007年第2期67-69,72,共4页Stroke and Nervous Diseases

基　　金：国家自然科学基金资助项目(NO.30270490);重庆市科学计划项目(NO.7452)

摘　　要：目的 探讨胎鼠皮质神经元分离和培养的方法,并对分离得到的细胞进行鉴定.方法 分离17 d胎鼠大脑皮质,切碎后经0.3%胰蛋白酶和DNaseI消化得到细胞悬液,用含N1添加剂和10%胎牛血清的MEM培养基进行培养,相差显微镜下观察神经元的生长情况.培养第3和6 d以微管相关蛋白-2（MAP-2）和神经元核心抗原（NeuN）为标志物,用免疫细胞化学的方法对分离培养的细胞进行鉴定.结果 培养第5～6 h时神经元已贴壁,长出数个小突起,培养第3 d可见突起增多、延长,培养第6 d细胞突起明显延长,相邻细胞的突起交错形成网络 培养第3 d MAP2免疫反应阳性细胞率（87.5±3.5）%,与培养第6 d的阳性细胞率（90.2±5.1）%无明显差异（P＞0.05） 培养第3 d NeuN免疫反应阳性细胞率（92.4±2.7）%,与培养第6 d的阳性细胞率（88.7±4.6）%无明显差异（P＞0.05）.结论 本方法分离培养的神经元纯度高,在含N1的完全培养基中培养神经元存活状态良好,未见胶质细胞明显增生.Objective To establish the method of isolating and culturing neurons from the cerebral cortex of embryonic rats and to identificate them. Methods The cortex of E17 rats was isolated and minced. The cell suspension was harvested by 0. 3M trypsin and DNaseI and incubated in MEM medium added with N1 medium supplyrnent and 10% fetal bovine serum. The growth of the neurons was observed with contrast phase microscope. The cells were identificated after immunostaining with MAP2 and NeuN on the 3rd day and 6th day after incubation. Results The neurons deformed and had processes at 5-6 hours. The neurons grew well. The processes prolonged on the 3rd day and cross linked on the 6th day after incubation. The ratio of MAP2- reactive neurons was （87. 5±3.5）M on the 3rd day and （90. 2± 5. 1）M on the 6th day after incubation. The ratio of NeuN-reactive neurons was （92. 4± 2. 7） % on the 3rd day and （88. 7 ± 4. 6） % on the 6th day after incubation. The ratio of MAP2-reactive or NeuN-reactive neurons did not decrease significantly on the 6th day vs 3rd day after incubation（P〉0. 05）. Conclusions The purity of neurons isolated with this method is high and the neurons grow well in MEM medium added with N1 medium supplyrnent, moreover the gliocytes do not proliferate significantly with this method. We think it is an effective method in isolating and culturing neurons.

关 键 词：大脑皮质 神经元 胚胎 微管相关蛋白 

分 类 号：R741[医药卫生—神经病学与精神病学]