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目的 研究逆转录病毒介导多药耐药基因 MDR1的转移与表达。方法 用 MDR病毒转导人类白血病细胞K5 6 2和 NB4,获得耐药细胞系 ;外源性 MDR1基因的转移和表达用聚合酶链反应 (PCR)、流式细胞术 (FCM)和半固体集落培养法分析。结果 MDR病毒转导使白血病细胞获得经典多药耐药表型 ,78.0 %～ 98.7%的细胞表达 P-糖蛋白 ;PCR分析证实耐药细胞整合有 MDR原病毒 ,并共表达全长和异常剪切 MDR1转录本。以 K5 6 2细胞为模型 ,FCM和集落培养法发现共培养法的基因转导率 (6 7.9%～72 .5 % )明显高于上清法 (33.1%～ 46 .8% ) ,加入生长因子可提高基因转导率约 2 3%。结论 逆转录病毒可介导MDR1基因在髓系白血病细胞进行有效的转移与表达 ;MDR1基因可作为选择性标志用于基因治疗
Objective To study the transcription and expression of retrovirus-mediated multidrug resistance gene MDR1. Methods MDR virus was used to transduce human leukemia cells K562 and NB4 to obtain drug-resistant cell lines. The exogenous MDR1 gene was transferred and expressed by polymerase chain reaction (PCR), flow cytometry (FCM) and semi-solid colony Cultivation analysis. Results MDR virus transduced leukemia cells into a classic multidrug resistance phenotype, and 78.0% ~ 98.7% of the cells expressed P-glycoprotein. PCR analysis confirmed that the resistant cells were integrated with the MDR provirus and coexpressed full length and abnormal scissors Cut MDR1 transcript. K562 cells as a model, FCM and colony culture method found that the co-culture of gene transduction rate (6 7.9% ~ 72.5%) was significantly higher than the supernatant method (33.1% ~ 46.8%), adding growth Factors can increase gene transfer about 23%. Conclusion The retrovirus can mediate the efficient transfer and expression of MDR1 gene in myeloid leukemia cells. The MDR1 gene can be used as a selectable marker for gene therapy