摘要
目的 增强造血细胞对化疗药物的耐受性 ,探讨逆转录病毒介导的基因转移效率及耐药基因导入在造血细胞保护性基因治疗中的作用。方法 应用电穿孔基因转移法将逆转录病毒载体G1Na MGMT IRES MDR1导入病毒包装细胞GP +E86及PA3 17,以VCR加压筛选后的阳性克隆上清感染小鼠骨髓细胞 ,应用PCR、Southernblot及流式细胞仪检测耐药基因MGMT及MDR1在细胞中的转移与表达。结果 加压筛选及乒乓效应使病毒滴度由 ( 0 .2～ 7.6)× 10 4CFU/mL提高到 ( 1.9～ 5 .7)× 10 6 CFU/mL ,从DNA、RNA及蛋白水平上分别证实了双耐药基因在小鼠骨髓细胞中获得了有效转移和表达 ,而转基因后的小鼠骨髓细胞对VCR、高三尖杉酯碱和BCNU的耐药性比未转导细胞分别提高了 5 .7、5 .0和 8.5倍。结论 双耐药基因成功导入小鼠骨髓细胞为肿瘤基因治疗的临床研究提供了实验基础。
Purpose To explore murine bone marrow cells transduced with drug resistance gene increase resistance to chemotherapy drugs. Methods The retroviral vector G1Na-MGMT-IRES-MDR1 was transduced into the packaging cell lines GP + E86 and PA317. After using VCR for cloning selection, murine bone marrow cells were transfected repeatedly with supernatant of retrovirus containing 06-methylguanine-DNA-methyltransferase(MGMT) and multidrug resistance (MDR1) cDNA. Southern blot, RT - PCR and FACS method analyses were used to show whether the genes were efficiently transduced into target cells. Results After using VCR for cloning selection and ping-pong effect, the highest titer was up to (1.9 - 5.7) × 106 CFU/mL from (0.2 - 7.6) x× 104 CFU/mL. The MGMT and MDR1 genes have been integrated into the genomic cDNA of murine bone marrow cells and expressed efficiently. The transgenes recipient cell confered 5.7, 5.0 and 8.5 folds stronger resistance to VCR, Homoharringtonine and BCNU. Conclusions Retroviral vector mediated double drug resistance genes transfer of murine bone marrow cells could confer the resistance of transgene cells to combination chemotherapy. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.
出处
《复旦学报（医学版）》
EI
CAS
CSCD
北大核心
2002年第1期1-5,共5页
Fudan University Journal of Medical Sciences
