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目的 构建携带多药耐药mdr1全长基因的真核表达载体并研究其在人肝癌细胞HepG2 中的表达。方法 双酶切质粒pHaMDR1,获取含mdr1全长cDNA片断,将此片段定向克隆到真核表达载体PCI neo多克隆位点,经脂质体法转染HepG2细胞,G418筛选稳定的细胞系HepG2/mdr1,PCR检测mdr1 特异片段,RT PCR 检测HepG2/mdr1细胞mdr1 mRNA表达,流式细胞仪检测HepG2/mdr1 细胞P gp的含量。结果 成功构建携带mdr1全长基因的真核表达载体,并在HepG2 细胞中表达,形成稳定的细胞系HepG2/mdr1,其mdr1 mRNA及P gp的含量较未转染该载体的HepG2细胞显著增加。结论 用真核表达载体将mdr1 全长基因导入人肝癌细胞HepG2能够建立高效、稳定的多药耐药细胞系,为进一步研究多药耐药机理提供理想的细胞模型。
Objective To construct eukaryotic expression vector carrying multidrug resistance mdr1 gene and investigate its expression in HepG2 cells. Methods The full-length cDNA fragment containing mdr1 was obtained by digesting the plasmid pHaMDR1. The fragment was cloned into the eukaryotic expression vector PCI neo multi-cloning site and transfected into HepG2 cells by lipofectamine. The stable cell line HepG2 / The mdr1 specific fragment was detected by mdr1 and mdr1 mRNA expression in HepG2 / mdr1 cells by RT PCR. The content of P gp in HepG2 / mdr1 cells was detected by flow cytometry. Results The eukaryotic expression vector carrying mdr1 full length gene was successfully constructed and expressed in HepG2 cells. The stable cell line HepG2 / mdr1 was constructed, and the mdr1 mRNA and P gp content were significantly increased compared with HepG2 cells without transfection. Conclusion The eukaryotic expression vector mdr1 full-length gene into human hepatoma HepG2 cells can establish efficient and stable multidrug resistance cell lines, and provide an ideal cell model for further study of multidrug resistance mechanism.