含MDR基因的人乳腺癌MCF 7/pZMDR多药耐药细胞株的构建

目的：肿瘤细胞的多药抗药性（ＭＤＲ）是化疗失败的主要原因之一，Ｐ－糖蛋白高表达是ＭＤＲ的主要机制，逆转ＭＤＲ成为肿瘤治疗亟待解决的问题。目前用于研究抗药性和筛选逆转抗药性药物的模型较少，本实验拟构建多药抗药性细胞株。方法：采用基因工程技术，重组ＭＤＲ１基因ｃＤＮＡ于逆转录病毒载体ｐＺＩＲ－ＮｅｏＳＶ（Ｘ）的克隆位点，并用脂染胺（ｌｉｐｏｆｅｃｔＡＭＩＮＥ）介导的ＤＮＡ转移技术，将其转入包装细胞ＰＡ３１７中，收集含病毒子的培养上清液感染对药物敏感的人乳癌细胞株ＭＣＦ－７，经筛选培养基筛选，ＰＣＲ、免疫组化及阿霉素在细胞内的分布等实验。结果：含ＭＤＲ基因的逆转录病毒载体ｐＺＭＤＲ的构建方法证明是正确的，ＭＤＲ１ｃＤＮＡ已整合在染色体基因组中并表达Ｐ－糖蛋白。结论：建立的ＭＣＦ－７／ｐＺＭＤＲ细胞为一特异表达Ｐ－糖蛋白的多药抗药性转基因细胞株

The construction of multidrug resistance cell lineMCF 7/pZMDR of human breast cances

Purpose: Clinical resistance to chemotherapeutic drugs is a major hinderance in the ohemotherapy of cancer. The main mechanism of MDR is the high expression of P-glycoprotein. The reversal of MDR is the problem that should hurry up resolvent. The drug-resistant cell line is an important model to study multidrug resistance in vitro. Methods: We have constructed a retrovirus expression vector pZMDR that carries MDR1 cDNA and was inserted into the pZIP-NeoSV(X) vector. The pZMDR was transfected into PA317 packaging cells by using LipofectAMINE. The medium was collected from G418- resistant PA317 cells (called PA317/pZMDR) and infected drug-sensitive human breast cancer cells (MCF-7). The clones resistant to G418 (MCF-7/pZMDR) were selected. Results: PCR, immunocytochemistry and accumulation of intracellular Adriamycin by microfluorescence showed that this construction was correct, MDR1 cDNA was intercalated genomic and express P-glycoprotein. Conclusion: MCF-7/pZMDR cells are MDR cells which express P-glycoprotein.