转mdrl基因诱导C工K细胞耐药并保持肿瘤杀伤活性杨永红李惠芳石永进.doc

转mdrl基因诱导CIK细胞耐药并保持肿瘤杀伤活性 杨永红 李惠芳 石永进 王逸群 张英 王奕嘉 朱平 [摘要] 目的 将多药耐药基因(mdr1)转入细胞因子诱导的杀伤(cytokine-induced killer，CIK)细胞，观察其是否产生耐药性并且保持肿瘤杀伤活性。方法 用IFN-γ，CD3单抗，IL-2，IL-1等细胞因子体外诱导外周血单个核细胞获得CIK细胞。采用电穿孔方法将mdr1基因表达质粒pHamdr，转入CⅨ细胞。转染后72h提取细胞总RNA，DNA酶消化残留质粒DNA后进行RT-PCR，鉴定mdr1基冈表达；流式细胞仪检测细胞膜耐药蛋白(P-gp)表达。四唑蓝比色法(MTT)检测转基因后CIK细胞对阿霉素和秋水仙碱的耐药性；同时检测转染前后CIK细胞对MCF7细胞(人类乳腺癌细胞系)的杀伤活性变化。结果 转染mdr1后的CIK细胞mdr1 mRNA阳性，P-gp阳性的CIK细胞为21％～37％(平均27％)。转染后CIK细胞获得了多药耐药性，对阿霉素的IC50值较转染前升高了22．3~45．8倍，对秋水仙碱的IC50值升高了6．7～11．35倍。转染前后CIK细胞对MCF7肿瘤细胞的杀伤活性无明显变化(P 0．05)。结论 CIK细胞转入mdrl基因后获得了多药耐药性，转染后的CIK细胞仍然保持原有的肿瘤细胞杀伤活性。 [关键词] 基因，mdr1； 抗药性，多药； 杀伤细胞，细胞因子诱导； 基因转染 CIK cells acquired multidrug resistance and maintained cytotoxic aetivity to tumor cells after mdrl gene transfection YalGYong-hong，LI Hui-fang，SHI Yong-jin，WANGYi-qun，ZHANGYing，WANG Yi-jia，ZHU Ping. Beijng Univensity Finst Hospital，Beijing 100034， China [Abstract] Objective To investigate whether the cytokine-induced kilier (CIK ) cells could acquire multidrug resistance and maintain the original cytotoxic activity after multidrug resistance (mdr1) genes transfection. Methods CIK cells were generated from peripheral blood cultured with IFN-γ，CD3 monoclonal antibody，IL-2，IL-1 and transfected with a plasmid (pHamdr) containing human mdrl gene via electroporation，RT-PCR method was used to assay mRNA expression of mdrl gene in transfectec CIK cells，flow cytometry with anti-P-gp monoclonal antibody to detect P-glycoprotein (P-gp) expression on CIK cells membrane，and MTT assay to compare both the multidrug resistance to doxorubicin and colchicines and cytotoxic activity to human mammary cancel cell line MCF7 between transfected and non-transfected CIK cells．Result mdrl expressmn was detected in the transfected CIK cells. There was a strong expression of P-gp on the transfected CIK cells and the percentages of P-gp positive cells were 21％～37％(average 27％). The IC50 of transfected CIKcells to doxorubicin was 22．3～45．8 times a