脊髓腹侧神经元的纯化培养与鉴定  

Purification and classified identification of neurons from ventral spinal cord in vitro

作　　者：文灿[1,2] 黄河[3] 王晗知[1] 张艳玲[1] 梁亚杰[1] 郭强[1] 苏炳银[1,4] 

机构地区：[1]第三军医大学基础医学部神经生物学教研室,重庆400038 [2]第三军医大学基础医学部外科应用解剖与手术学教研室,重庆400038 [3]第三军医大学新桥医院麻醉科,重庆400037 [4]成都医学院组织胚胎与神经生物学教研室,四川成都610081

出　　处：《局解手术学杂志》2010年第6期455-458,共4页Journal of Regional Anatomy and Operative Surgery

基　　金：重庆市自然科学基金2009BB5150;四川省教育厅重大培育项目;四川省科技厅项目(NO.2008JY0081)

摘　　要：目的探讨胚胎大鼠脊髓腹侧组织神经元的分离、纯化和培养方法,并予以分类鉴定和测定其纯度。方法取胚胎大鼠脊髓腹侧组织分离成细胞悬液,经差速贴壁后在无血清条件培养基里培养,采用免疫细胞化学双标染色法对培养盖片上的细胞予以鉴定、分类,结合Hoechst荧光染核,计数各细胞成分的含量。结果细胞贴壁生长好,神经元占87.8%,其中,运动神经元达70.9%,星形胶质细胞占7.5%,NF200和GFAP染色皆为阴性的细胞占4.7%。结论差速贴壁接种法结合无血清条件培养基培养脊髓腹侧组织能有效抑制星形胶质细胞的快速增殖,能培养出高纯度的神经元,尤其是脊髓运动神经元。Objective To explore the method of isolation , culture and purification of neurons from ventral spinal cord tissues in embry- onic rats in order to do classified identification and to fix purity coefficients. Methods Ventral spinal cord tissues of embryonic rats were dissected and cell suspensions were made. Cells were cultured in serum - free medium after treating by differential adhesion method and the classified identification was made by double immunoeytoehemistry labeling stain. In addition, the nucleuses were stained by Hoeehst and the contents of the cells were counted. Results Cells adhered and grew well. Immunocytochemistry revealed that the neuronal purity was up to 87.8% with the purity of spinal motor neurons, astroeyte and negative cells stained by NF200 and GFAP being 70.9%, 7.5% and 4.7% respectively. Conclusion The rapid proliferation of astrocyte could be inhibited efficiently and neurons with high purity, especially spinal cord neurons could be got by method of differential adhesion associated with serum - free culture medium.

关 键 词：神经元 胆碱乙酰转移酶 神经微丝蛋白 胶质纤维酸性蛋白 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] Q343.6[医药卫生—基础医学]