胶质瘤中脑肿瘤干细胞的分离、培养和鉴定  

Isolation,culture,and identification of brain tumor stem cell in gliomas

作　　者：王存祖[1] 赵鹏[1] 尤永平[1] 陆小明[1] 陈云祥[1] 傅震[1] 鲁艾林[1] 刘宁[1] 

机构地区：[1]南京医科大学第一附属医院神经外科,210029

出　　处：《江苏医药》2009年第1期8-11,F0002,共5页Jiangsu Medical Journal

基　　金：江苏省自然科学基金(BK2005156)

摘　　要：目的分离、培养和鉴定胶质瘤中脑肿瘤干细胞(BTSC),探讨CD133作为BTSC特异性标记,用于BTSC分离的可行性。方法9例胶质瘤临床标本,胶质瘤C6和U87细胞系,用直接培养和磁珠选择CD133+细胞二种方法分离BTSC。参照NSC培养条件,进行体外培养。观察细胞生长情况,利用免疫荧光染色检测细胞标记,包括Nestin,神经元特异性烯醇化酶(NSE),胶质纤维酸性蛋白(GFAP)等;诱导细胞分化,验证该种细胞是否具有干细胞特性。结果9例临床标本中,有2例儿童恶性胶质瘤在培养过程中出现神经球,在成人胶质瘤中均未出现。C6和U87细胞中,均出现比较典型的神经球,二种分离方法结果相同。神经球细胞具有增殖能力,体外培养可以不断增大。标记检测显示这种细胞Nestin阳性,而NSE和GFAP阴性。经诱导后,可以分化为形态与来源细胞相似的细胞,能够表达NSE和GFAP等多种表型细胞标记。结论在胶质瘤中,存在具有NSC特性的干细胞,即BTSC。CD133是BTSC重要的表面标记,可以用于BTSC的分离。在胶质瘤细胞系中CD133+细胞是BTSC。Objective To investigate the feasibility of isolation of brain tumor stem cell （BTSC） in gliomas by labeling with CD133. Methods Nine human gtiomas specimens, two glioma cell lines （C6 and U87）, were involved in this study. The methods of isolation included primary culture and CD133^＋ cells culture after magnetic cell sorting by CD133 cell isolation kit. Cells cultures were performed under the condition of neuron specific enolase （NSE）. The BTSC was identified by immunocytochemical staining of neurospheres by detected the types of molecular markers of nestin, NSE, glial fibrillary acidic protein （GFAP）, and by differentiation tests as well. Results Classical neurospheres formed in vitro by normal NSC were detected in two glioma specimens cultures established from pediatric malignant gliomas. They were medulloblastoma and high-grade glioma. The neurospheres derived from high-grade glioma could have. long-term cultures, while from medulloblastoma could not. Conversely, clone formation was never observed in cultures from adult gliomas. The neurospheres were also detected in C6 and U87 cell lines either by primary culture or by CD133^＋ cells culture. The neurospheres could proliferate and increase in their diameters. The labeling test showed nestin positive, but NSE and GFAP negative. The induced cells ceuld defferentiate into the cells which had the same from as the original cells and expressed NSE and GFAP. Conclusion The results show that gliomas contain neural stem-like cells, which are BTSC. The BTSC can be exclusively isolated with the cell fraction expressing the neural stem cell surface marker CD133.

关 键 词：胶质瘤 脑肿瘤干细胞 CD133 

分 类 号：R739[医药卫生—肿瘤]