胃癌耐药细胞株OCUM-2M/VP16的建立及其耐药机制  

Establishment of VP16-resistant gastric cell line and its multi-drug resistance mechanism

作　　者：邱红[1] 丁方勇[2] 熊慧华[1] 张明生[1] 李瑞超[3] 陈元[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肿瘤科,湖北省武汉市430030 [2]武汉市急救中心,湖北省武汉市430022 [3]华中科技大学同济医学院附属同济医院综合科,湖北省武汉市430030

出　　处：《世界华人消化杂志》2009年第18期1809-1814,共6页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30801369;日中医学协会笹川医学奖学金资助项目;No.2007-2008[30]~~

摘　　要：目的:建立耐VP16的胃癌耐药细胞株,并初步探讨细胞发生VP16耐药的可能机制.方法:采用药物浓度梯度递增法诱导建立OCUM-2M/VP16耐药株.细胞计数法绘制细胞生长曲线,计算细胞倍增时间.MTT法检测细胞对6种化疗药物的IC50.流式细胞仪分析细胞周期分布.逆转录多聚酶链反应检测caspase-3,P53,DAPK-1,DAPK-2,DAPK-3,Bcl-2,ERCC-1,MDR-1及MRP基因mRNA的表达.结果:OCUM-2M/VP16成功实现了对VP16的耐药,耐药指数为40.53;其增殖速率及细胞倍增时间明显低于OCUM-2M(细胞倍增时间:22.96±0.96h vs 30.29±2.55h,P<0.01).OCUM-2M/VP16对Sn38、奥沙利铂及吉西他滨发生了交叉耐药,但对5-FU及紫杉醇敏感性与亲代类似.OCUM-2M/VP16凋亡相关基因DAPK-2,DAPK-3和Bcl-2表达下调(OCUM-2M表达值为:0.61、0.79及0.81,OCUM-2M/VP16为,0.24、0.45、0.44,P<0.01),耐药相关基因ERCC-1和MDR-1表达上调(OCUM-2M为0.53及0.20,OCUM-2M/VP16则为0.84及0.41,P<0.01).caspase-3,P53,DAPK-1及MRP表达无变化.结论:成功构建胃癌耐药细胞株OCUM-2M/VP16,该细胞株具有交叉耐药特性,耐药机制涉及凋亡、耐药等多个基因表达变化.AIM： To establish the VP16 resistant gastric cell line and to explore its potential multi-drug resistant mechanism. METHODS： We used the drug concentration step-elevation method to establish VP16 resistant sub-line of gastric cell line OCUM-2M. Growth curves of cells were delineated and cell doubling times of cells were calculated using cell-counting methods. IC50 of chemotherapy drugs in two cell lines were determined by MTT methods. Cell cycle distributions were tested by FCM analysis. mRNA expression levels of caspase-3, P53, DAPK-1, DAPK-2, DAPK-3, Bcl-2, ERCC-1, MDR-1, and MRP were determined by RT-PCR. RESULTS： OCUM-2M was a successful drug-resistant cell line, and the resistance index to VP16 was 40.53. The cell doubling time of OCUM-2M/VP16 was 30.29 ± 2.55 h, while that of parental cell line OCUM-2M was 22.96 ± 0.96 h （P 〈 0.01）. The cross-drug-resistance of Sn38, oxaliplatin, and gemcitabine in OCUM-2M/VP16 was observed, while chemo-sensitivity of 5-FU and paclitaxol in OCUM-2M/VP16 remained the same with OCUM-2M. In OCUM-2M/VP16, the mean expression levels of apoptosis related genes, DAPK-2, DAPK-3 and Bcl-2 were respectively 0.24, 0.45, and 0.44, which were lower than OCUM-2M （0.61, 0.79 and 0.81）. The expression levels of drug-resistance related genes ERCC-1 and MDR-1 in OCUM-2M/VP16 were respectively 0.84 and 0.41, which were higher than OCUM-2M （0.53 and 0.20, P 〈 0.01）. The expression levels of caspase-3, P53, DAPK-1 and MRP had no significant change in the two cell lines. CONCLUSION： OCUM-2M/VP16 is a successful VP16-resistant gastric cell line with cross-resistance ability, and the expression level changes of apoptosis and drug resistance related genes might contribute to drug-resistance in OCUM-2M/VP16.

关 键 词：鬼臼乙叉甙 胃癌 多药耐药 凋亡 

分 类 号：R735.2[医药卫生—肿瘤]