逆转录病毒介导耐药基因的高效表达

Efficient Expression of Drug Resistance Gene Mediated by Retrovirus

目的 建立多药耐药基因MDR1的逆转录病毒转移系统。方法 脂质体转染法将携带MDR1基因的逆转录病毒载体HaMDR导入单向型包装细胞GP +E86 ;用获得的单向型病毒重复转导双嗜型包装细胞GP +envAm12 ,经秋水仙碱加压选择获得高滴度的病毒生产细胞Am12 /HaM DR ;MDR1基因的转移和表达用聚合酶链反应 (PCR)、MTT比色法和流式细胞术 (FCM )分析。结果 单向型与双嗜型病毒生产细胞的滴度分别为 6 2× 10 5和 9 0× 10 5CFU/ml;PCR分析证实生产细胞有MDR1基因整合并稳定表达 ,但同时存在异常剪接的转录本 ;MTT比色法和FCM分析显示MDR1基因转移细胞的耐药程度提高 12～ 2 6 7倍 ,P 糖蛋白表达率达 97%～ 99%。结论 逆转录病毒可有效介导MDR1基因转移和表达 ,为导入造血干 /祖细胞后进行根治性化疗奠定基础。

Objective To establish a retrovirus mediated gene transferring system for multi drug resistance gene MDR1. Methods Using liposome,GP+E86 ecotropic packaging cells were transfected with the retroviral vector HaMDR carrying human MDR1 gene.Then,the high titer amphotropic retroviral producer cells,named Am12/HaMDR,were generated via infecting the GP+envAm12 packaging cells by ecotropic virus repeatedly and selecting with colchicine up to 400μg/L.The transfer and expression of the exogenous MDR1 gene was probed by polymerase chain reaction(PCR),MTT colorimetric assay and flow cytometry(FCM).Results Viruses in supernatants were titrated to 6 2×10 5CFU/ml for GP+E86/HaMDR cells and 9 0×10 5CFU/ml for Am12/HaMDR cells.Confirmed by PCR assay,transfection of HaMDR vector resulted in both integration and overexpression of MDR1 gene in both producer cells,in which aberrant spliced transcripts were also noted.Moreover,the MDR1 transgene was effectively translated into P glycoprotein in 97%～99% of resistant cells,and resulted in a 12 to 267 fold drug resistance in comparison with parental cells,analyzed by FCM and MTT assay.Conclusion The transfer and expression of MDR1 could be efficiently mediated by retrovirus,suggesting the potential utility of the HaMDR vector in protecting hematopoietic stem/progenitor cells from ablative chemotherapy.