胶质瘤干细胞对替莫唑胺敏感性的研究

Analysis of the sensitivity of glioma stem cells to temozolomide

目的探讨胶质瘤干细胞（GSC）对替莫唑胺（TMZ）的敏感性及耐药机制。方法新鲜多形性胶质母细胞瘤（GBM）标本培养后获得GSC。免疫荧光技术检测未分化GSC的CD133及分化生长GSC的GFAP的表达，MTS法检测对替莫唑胺的敏感性，流式细胞技术对CD133阳性细胞的比例进行定量，荧光标记的甲基化特异性PCR分析MGMT启动子区域的甲基化状态，Western blot检测抑癌基因PTEN的表达。结果（1）5例GBM标本中成功获得GSC，符合肿瘤干细胞定义。（2）5个GSC细胞株多数对TMZ不敏感。其中，T509的半数抑制浓度（IC50）为22．3μmol／L（敏感），T411的IC50为286．3μmol／L（中度敏感），其余3个细胞株T402，T405及T509的IC50皆大于1000μmol／L（不敏感）。（3）CD133阳性细胞比例大于10％的GSC细胞株对TMZ不敏感。（4）MGMT启动子区域呈去甲基化状态的GSC对TMZ不敏感或仅为中度敏感。（5）5个GSC细胞株中，PTEN表达水平差异大，与GSC对TMZ的敏感性无明显关联。结论GSC对TMZ普遍耐药，与MGMT启动子区域甲基化状态及CD133阳性细胞有关，而与PTEN蛋白表达水平无明显关联。

Objective To investigate the sensitivity of glioma stem cells （GSC） to temozolomide （TMZ）. Methods Five GSC lines were successfully established from fresh glioma tissues. Immunofluoresenee was employed to detect the expression of CD133 in undifferentiated GSC and glial fibrillary acid protein （GFAP） in differentiated GSC. The sensitivity of GSC was determined by using MTS. The percentage of CD133 - positive cells was measured with flow cytometry. The methylation of MGMT promoter region in GSC was determined by using fluorescent methylation specific PCR （F - MSP）. Western blotting was used to determine the protein level of PTEN in GSC. Results （ 1 ） The 5 established GSC lines demonstrated characteristics of cancer stem cells. （2） GSC lines were generally resistant to TMZ. The ICs0 of TMZ was 22. 3 μmol/L for T509, 286. 3 μmol/L for T411, and more than 1000 μmol/L for T402, T405 and T511, respectively. （3） GSC lines with more than 10% CD133 -positive cells were more resistant to TMZ. （4） GSC lines with unmethylated MGMT promoter region were resistant or intermediately sensitive to TMZ. （5） The protein level of PTEN was different in the 5 GSC fines. No relationship between PTEN level and TMZ sensitivity was found. Conclusion A majority of GSC is resistant to TMZ, which is associated with the methylation status of MGMT promoter and CD133 - positive cell population.