中文摘要：

目的探讨人脑胶质瘤SHG-44细胞O（6）-甲基鸟嘌呤DNA甲基转移酶（MGMT）基因启动子甲基化状态与其蛋白表达以及细胞对烷化剂药物耐药性的相关性。方法取处于对数生长期的人脑胶质瘤SHG-44和U251细胞，分别加入5-氮-2＇脱氧胞苷（5-Aza-CdR）培养6d后收集细胞，同时以未加5-Aza-CdR的正常培养细胞作为对照。提取细胞基因组DNA，采用甲基化特异性PCR（MSP）检测MGMT基因的甲基化状态，并利用蛋白质印迹方法检测MGMT蛋白的表达。将收集的细胞分为3组，分别加入浓度为125、100、75、50、25、15、10μg／ml的尼莫司汀（ACNU）和50、25、15、10、5、2．5、1μg/ml的替莫唑胺（TMZ）以及等量的完全培养液（阴性对照），采用噻唑蓝（MTT）法分别检测细胞对烷化剂药物的敏感性，以细胞存活50％时所对应的药物浓度（IC50值）作为衡量细胞对药物敏感性的指标，实验重复3次。结果正常培养的SHG-44细胞MGMT基因启动子呈甲基化状态，MGMT蛋白表达缺失，对ACNU和TMZ的IC50值分别为30、11μg／ml，表现为对烷化剂药物敏感；用5-Aza-CdR处理后，SHG-44细胞MGMT基因启动子成功脱甲基化，MGMT蛋白恢复表达，其对ACNU和TMZ的IC50值分别升高了2．5和3．1倍（均P〈0．05），对烷化剂药物的敏感性发生逆转。而正常培养和5-Aza-CdR处理的U251细胞MGMT基因启动子均呈未甲基化状态，都能表达MGMT蛋白，并且均表现为对ACNU和TMZ烷化剂药物耐药。结论MGMT基因甲基化状态能稳定地反映细胞诱导MGMT蛋白表达的能力，并有可能成为预测肿瘤组织对烷化剂化疗药物敏感性的分子标记。

英文摘要：

Objective To explore the MGMT gene methylation status and its protein expression as well as cells to alkylating agent drug resistance related to each other in glioblastoma cell line SHG-44 cells.Methods In the logarithmic phase of the human glioblastoma cell line SHG-44 and U251 cells were collected and treated with 5-Aza-CdR for 6 days. The cells untreated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the SHG-44 and U251 cells, using methylation-specific PCR （MSP） to detect promoter CpG island methylation status of the MGMT gene. The expression of MGMT protein was detected by Western blotting. The SHG-44 and U251 cells were divided into 3 groups, and treated with ACNU of different concentrations （125, 100, 75, 50, 25, 15, and 10 μg/ml, respectively）, TMZ of different concentrations （50, 25, 15, 10, 5, 2.5, and 1 μg/ml, respectively）, and the same volume of complete culture medium （negative control）, respectively. The sensitivity of the SHG-44 and U251 cells to alkylating agent drug was analyzed by MTT method, the drug concentration corresponding to 50% cell survival （IC50 value） was set as a measurable indicators. Each experiment was repeated three times.Results With promoter CpG island methylation of the MGMT gene, no MGMT protein was expressed in the normal SHG-44 cells, and its IC50 values to ACNU and TMZ were 30 μg/ml and 11 μg/ml respectively, showing sensitive to alkylating agents ACNU and TMZ. After being treated with 5-Aza-CdR, the SHG-44 cells restored expressing MGMT protein following its MGMT gene successful demethylation, and its ICso values to ACNU and TMZ were increased by 2.5 and 3.1 times respectively （both P 〈 0.05）, showing resistive to alkylating agents ACNU and TMZ. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal U251 cells and the U251 cells treated with 5-Aza-CdR, and both showing resistive to alkylating agents ACNU and TMZ. Conclusion The promoter CpG island methyl