携带反义MDR1基因的逆转录病毒载体的构建和表达

Construction and expression of retroviral vector carrying antisense MDR1

目的 建立逆转录病毒介导的反义多药耐药MDR1基因转移体系 ,探讨反义RNA封闭白血病耐药细胞中MDR1基因表达的能力。方法 逆转录 聚合酶链反应 (RT PCR)法从白血病耐药细胞株HL 6 0 /VCRmRNA中扩增出含有启始密码子ATG的 5 2 4bp的MDR1基因cDNA片段 ,反向插入逆转录病毒载体 pLXSN ,通过脂质体转和乒乓感染单、双噬型包装细胞GP +E86和GP +envAM 12 ,以双嗜型病毒上清感染白血病耐药细胞K5 6 2 /MDR ,半定量RT PCR和流式细胞术(FCM)分析MDR1基因转录和P 糖蛋白 (P gp)的表达。 结果 酶切、PCR和DNA测序证实反义MDR1基因重组逆转录病毒载体 pLASSN构建正确 ;双嗜型病毒生产细胞的滴度为 1 3× 10 5CFU/ml,巢式PCR和补救分析均未检测到辅助病毒存在 ;PCR和RT PCR分析证实病毒生产细胞和反义修饰的耐药细胞K5 6 2MDR/LASSN中均有反义MDR1基因整合和MDR1基因反义RNA的转录 ;FCM分析K5 6 2MDR/LASSN细胞中P gp表达强度和表达率比对照细胞均有明显下降。 结论 逆转录病毒介导的反义基因转移系统安全有效 。

Objective To establish an antisense MDR1 gene transfer system mediated by retroviral vector and study the inhibitory function of MDR1 antisense RNA on the MDR1 expression in drug resistant leukemic cells.Methods A 524bp cDNA fragment of MDR1 containing the translation start codon ATG was amplified from drug resistant cell line HL60/VCR by high fidelity RT PCR.The recombinant retrovial vector carrying antisense MDR1 gene was then constructed into pLXSN and both ecotropic,amphotropic retroviral producer cells were developed by liposome mediated transfection and cross infection.The transcription of MDR1 gene and expression of P gp in K562/MDR cells transducted with antisense gene were determined by RT PCR and flow cytometry (FCM).Results pLASSN was confered by restriction enzyme digestion,PCR and DNA sequencing analysis.The amphotropic viruses in supernatants were titrated to 1 3×10 5CFU/ml.No helper virus could be found by both nested PCR and rescue assay.Confirmed by PCR and RT PCR assay,transfection of pLASSN vector has resulted in intergration and expression of antisense MDR1 in both producer cells and K562MDR/LASSN cells.Compared with control cells,both the frequency and intensity of P gp expression were markedly reduced in antisense modified cells.Conclusion The efficiency and safety of the antisense gene transfer system may provide an experimental model in reversal research of mutidrug resistance of cancer.