替莫唑胺改变人胶质瘤细胞系U251耐药机制的体外研究  

Study on mechanism of temozolomide-resistant human glioma cell line U251/TR changed by temozolomide in vitro

作　　者：潘强[1] 杨学军[1] 高松[2] 纪延伟[3] 张文高[4] 李瑜[1] 王维[1] 董雪涛[1] 王华民[1] 

机构地区：[1]天津医科大学总医院神经外科,300052 [2]天津医科大学附属肿瘤医院胰腺肿瘤科 [3]山东省交通医院神经外科 [4]河北省沧州市中心医院神经外科

出　　处：《中国现代神经疾病杂志》2009年第6期583-588,共6页Chinese Journal of Contemporary Neurology and Neurosurgery

基　　金：国家自然科学基金资助项目(项目编号:30772228)

摘　　要：目的建立替莫唑胺耐药人胶质瘤细胞系,探讨其耐药性变化规律及耐药机制,以期为临床优化药物化疗方案提供理论依据。方法采用分步诱导法使人胶质瘤细胞系U251对替莫唑胺耐药,磺酰罗丹明B比色法检测细胞耐药指数和存活率;Western blotting法检测O^6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)表达水平,以及倍增替莫唑胺终浓度后MGMT表达水平。结果成功建立替莫唑胺耐药U251/TR细胞,在替莫唑胺初始诱导剂量0.25μg/ml、终浓度16.00μg/ml培养液中,U251/TR细胞半数抑制浓度为(220.87±2.34)μmol/L,约为U251细胞[(33.12±1.52)μmol/L]的7倍(t=-116.542,P=0.000),其耐药指数约为7;U251/TR细胞MGMT表达水平为(1.47±0.30),较U251细胞(0.19±0.03)明显升高(t=-20.230,P=0.000)。与溶媒对照组比较,经倍增替莫唑胺终浓度诱导的U251/TR细胞仍呈现增殖抑制现象,以体外培养第3天时最为明显(P=0.000);MGMT表达水平相应降低(均P=0.000),呈现自身克服耐药现象。结论采用分步诱导法可于体外成功建立替莫唑胺耐药人胶质瘤细胞系。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,替莫唑胺可以通过自身消耗MGMT而改变U251/TR细胞的耐药特性,发挥抗耐药作用。Objective To establish a drug-resistance cell line of human glioma with temozolomide （TMZ） and to explore the chemo-resistant evoluation in human glioma cell line U251 induced by TMZ, to investigate the mechanism of characteristics changed by TMZ in TMZ-resistant human glioma cell line U251/ TR and to provide experimental evidence for optimal TMZ therapy in malignant glioma treatment. Methods A TMZ-resistant human glioma cell line, U251/TR, was established by stepwise exposure of human glioma cell line U251 parental cells to TMZ. Sulfate-reducing bacteria （SRB） assay was used to calculate the resistance index （RI） and cell survival rate （SR）. Western blotting was used to detect O^6- methylguanine-DNA methyltransferase （MGMT） expression for the analysis of resistance mechanism and evoluation of TMZ-resistance in U251 cells during the induction procedure. Results A TMZ-resistant human glioma cell line, U251/TR, was developed after 8 months induction cultured with （0.25-16.00） μg/ml TMZ. IC50 in U251/TR cells [（220.87 ±2.34） μ mol/L] increased approximately 7 times compared to that in U251 ceils [（33.12 ±1.52） μmol/L; t = - 116.542, P = 0.000]. The MGMT expression was significantly increased in U251/TR cells （t =- 20.230, P = 0.000）. The growth of U251/TR cells was able to be inhibited significantly by TMZ with double strength （32.00 μg/ml） from 3 to 5 d compared with dimethyl sulfoxide （DMSO） control group （P ≤ 0.01, for all） and MGMT expression was decreased accordingly after TMZ administration （P = 0.000, for all）. Conclusion A TMZ-resistant human glioma cell line, U251/TR （RI ≈ 7）, was established by stcpwise exposure of human glioma cell line U251 parental cells to TMZ. The primary mechanism of U251/TR cells resistant to TMZ is owing to increase the expression of MGMT. TMZ can overcome the TMZ-resistance itself by consuming MGMT steadily. It implies that metronomic TMZ-regimen can improve the chemo-sensitivity in TMZ-resistant glioma clinically.

关 键 词：替莫唑胺 脑肿瘤 神经胶质瘤 抗药性 肿瘤 O(6)-甲基鸟嘌呤-DNA甲基转 移酶 

分 类 号：R739.4[医药卫生—肿瘤]