神经上皮肿瘤中脑肿瘤干细胞的分离培养和原发性多药耐药机制分析  

Isolation,culture and primary multidrug resistances mechanism of brain tumor stem cells within neuroepithelial tumors

作　　者：毕长龙[1] 方加胜[1] 陈风华[1] 王延金[1] 罗湘颖[1] 

机构地区：[1]中南大学湘雅医院神经外科,湖南长沙410008

出　　处：《中国现代医学杂志》2009年第2期172-176,180,共6页China Journal of Modern Medicine

基　　金：湖南省自然科学基金项目(No:055JJ30060)

摘　　要：目的探讨ABC家族转运体耐药基因MDR1、MRP1和DNA修复酶耐药基因MGMT在脑肿瘤干细胞原发性多药耐药中的作用机制。方法以CD133为脑肿瘤干细胞特异性标志物,利用免疫磁珠法分离30例人脑神经上皮肿瘤标本中的CD133+与CD133-细胞,并进行CD133+细胞的体外扩增培养,传代与鉴定。应用半定量RT-PCR法检测CD133+与CD133-细胞中耐药蛋白MDR1、MRP1、MGMT的活性表达情况。结果免疫磁珠法能有效的分选出脑肿瘤干细胞,该类细胞具有异常的增殖能力。MDR1、MRP1与MGMT耐药基因在CD133+细胞中高度表达,并与肿瘤的病理级别正相关,其总体水平分别为CD133-细胞的16.1、19.6及34.0倍。并非所有的脑肿瘤干细胞均有耐药基因的表达,MDR1、MRP1与MGMT的总体阳性表达率分别为76.7%、86.7%t和73.3%。ABC家族转运体耐药基因和DNA修复酶耐药基因在CD133+细胞中的表达有明显的相关性。结论神经上皮肿瘤中存在一定比例的CD133+脑肿瘤干细胞,免疫磁珠法能有效的分选出CD133+细胞。在异质性肿瘤中仅有小部分亚群细胞(CD133+脑肿瘤干细胞)具有原发耐药性,而ABC家族耐药蛋白和DNA修复酶在脑肿瘤干细胞中的共同过度表达是临床上脑肿瘤多药耐药的主要原因之一,脑肿瘤干细胞是神经上皮肿瘤原发性化疗耐药的根源和关键性治疗标靶。[Objective] To explore basic chemoresistance mechanism of ABC superfamily transporter and MGMT in these cells, observe the progress of proliferation for tumor stem ceils. [Methods] Brain tumor stem cells were isolated based on CD133 characteristic marker by MACS （magnetic activated cell sorting）. CD133^＋ cells were cultured and mechanically proliferated, passaged in serum-free medium. The drug-resistance proteins MDR1, MRP1, MGTM activity expression were analyzed between CDl33^＋ and CD133^- cells by RT-PCR. [Results] Brain tumor stem cells can be separated effectively by MACS and these cells have abnormally proliferating ability. CD133^＋ cells expressed high level of MDR1, MRP1 mRNA as well as higher level of MGMT mRNA than CD133^- ceils, the activities were totally increased to 16.1, 19.6, 34.0 times respectively and have positive correlation with pathological grades of tumors. MDR1, MRP1 and MGMT drug-resistance protein activity were not expressed in all the tumors, positive expressional rate was 76.7%, 86.7%, 73.3% respectively. There was statistic correlation between MDR1, MRP1 and MGMT protein activity expression in malignant tumors. [ Conclusion] Only a small proportion of cells in neuroepithelial tumors displayed strong capacity on tumor＇s resistances to chemotherapy. One of Neasous in these primary multidrug resistances of neuroepithelial tumors was probably contributed by brain tumor stem cells with higher expression of ABC superfamily transporter and DNA repair enzyme concomitantly. Brain tumor stem cells were the root of chemoresistances and key therapeutic target.

关 键 词：脑肿瘤干细胞 分离培养 多药耐药基因 免疫磁珠 CD133 

分 类 号：R739.4[医药卫生—肿瘤] R730.1[医药卫生—临床医学]