摘要
目的建立替莫唑胺耐药人脑胶质瘤细胞系,探讨其耐药机制,以期为临床优化药物化疗方案提供参考。方法通过体外分步诱导方法使人U251胶质瘤细胞对替莫唑胺产生耐药,噻唑蓝比色法检测耐药指数及细胞存活率;采用Western-Blot、逆转录-聚合酶链免疫(RT-PCR)、免疫荧光、免疫组化法检测O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)表达变化,分析细胞耐药机制。结果历经8个月成功建立了对替莫唑胺耐药的胶质瘤细胞U251/TR,在替莫唑胺初始浓度为0.25μg·mL-1,终浓度为16.00μg·mL-1培养液中,U251/TR细胞半数抑制浓度(IC50)为未经诱导的U251胶质瘤细胞的7倍(P=0.00),其耐药指数约为7.00;U251/TR细胞MGMT表达水平较未经诱导的U251胶质瘤细胞明显增加(P=0.00)。结论通过分步诱导方法于体外成功建立1株对替莫唑胺耐药的U251/TR细胞系。MGMT表达水平升高是导致U251/TR细胞对替莫唑胺耐药的主要机制。
Objective To establish a drug-resistance cell line of human glioma with temozolomide （ TMZ） ,investigate its resistance mechanisms, and provide experimental evidence for optimal TMZ therapy. Methods A TMZ-resistant human glioma cell line,U251/TR,was established by stepwise exposure of human parental U251 cells to TMZ. Resistance index and cell viability were accessed by MTT assay. Western-Blot,RT-PCR,immunohistochemistry and immunofluorescence were used to detect MGMT expression for the analysis of resistance mechanism. Results A TMZ-resistant human glioma cell line,U251/TR,was developed after 8 months of stepwise induction with 0. 25-16. 00 μg·mL^-1 TMZ. IC50 in U251/TR cells was approximately 7 times higher compared with that in U251 cells （P=0. 00 ）. The MGMT expression was significantly increased in U251/TR cells compared with that in parental U251 cells （P=0. 00） . Conclusion A TMZ-resistant human glioma cell line,U251/TR,was established by stepwise exposure of human parental U251 cells to TMZ. The primary mechanism of TMZ resistance is associated with increased activity of MGMT.
出处
《医药导报》
CAS
北大核心
2014年第9期1121-1125,共5页
Herald of Medicine
基金
国家自然科学基金资助项目(30772228)
