CYP3A5基因转染HL-60细胞介导耐药表型的研究

Transfection of HL-60 cells with CYP3A5 gene induces drug-resistant phenotype

目的探讨CYP3A5基因与白血病细胞多药耐药的关系。方法克隆CYP3A5基因全长cDNA,构建CYP3A5基因真核表达重组质粒,稳定转染HL60白血病细胞。采用四氮唑蓝法(MTT)测定化疗药的半数抑制量IC50值,采用流式细胞术(FCM)分析细胞周期及凋亡细胞百分比。结果空载质粒pcDNA3、重组质粒pcDNA3CYP3A5稳定转染HL60细胞。柔红霉素诱导后,HL60和HL60/pc细胞出现明显的凋亡峰,凋亡细胞比例分别为7.3%和6.3%;而HL60/CYP3A5细胞则无明显凋亡峰,凋亡细胞比例为1.2%。HL60/CYP3A5细胞与HL60、HL60/pc细胞相比,显著耐受柔红霉素、阿克拉霉素、长春新碱和三尖杉酯碱,耐药倍数分别为2.89,2.01,4.05和2.79倍(P<0.05);而对鬼臼噻吩甙则无明显耐受,耐药倍数为1.04倍。结论CYP3A5基因的转录直接导致白血病细胞对蒽环类抗生素及生物碱类药物耐药,而对表鬼臼毒素仍然敏感。

Objective To investigate if CYP3A5 gene is involved in the molecular mechanisms for multiple drug resistanee in leukemia cells. Methods A full length eDNA of CYP3A5 gene was cloned, and a recombinant eukaryotie expression plasmid was constructed, then stably transfeeted cell lines were established. Furthermore, the sensitivity of those cell lines to several antieaneer drugs were assessed by MTT and FCM assay. Results The recombinant plasmid was designated as pcDNA3-CYP3A5. Transfecting HL-60 cells（which didn＇t show transcript of CYP3A5 gene）with recombinant plasmid pcDNA3-CYP3A5 generated HL-60/CYP3A5 cell line, and transfeeting of HL-60 cells with the parental PcDNA3 vector served as control HL-60/pc cell line. Daunorubicin induced remarkable apoptosis peaks in HL-60 and HL-60/pc cells, while such effect did not occur in HL-60/CYP3A5 cells （apoptosis cell percentage were 7.3%, 6.3% and 1.2%, respectively）. Compared with HL-60 and HL-60/pc cells, HL-60/CYP3A5 cells were statistically significantly resistant to daunorublcin, aclacinomyein A, vincristine and harringtonine （resistance multiples were 2.89, 2.01, 4.05 and 2.79 times, respectively, P 〈 0.05 ）, however the sensitivity to teniposide didn＇t change （ resistance multiple was 1.04 times）. Conclusion Transcription of CYP3A5 gene in leukemia cells directly induces resistance to anthracyclines and alkaloids, however the cells are still sensitive to epipodophyllotoxins. Therefore, our findings confirmed a new mechanism of multidrug resistance.