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目的构建多药耐药基因(mdr1)启动子控制的胸苷激酶-胞嘧啶脱氨酶(CD-TK)双自杀基因真核表达载体,研究其对耐药白血病细胞K562／A02的靶向杀伤作用。方法利用分子克隆技术构建pcDNA3-mdr1 P-CD-TK真核表达载体,脂质体介导法转染K562、K562／A02细胞,通过G418筛选获得稳定转染的细胞株。用PCR、RT-PCR鉴定TK、CD基因的稳定转染与表达,加用不同浓度的丙氧鸟苷(GCV)、5-氟胞嘧啶(5-FC)培养4 d,用四甲基偶氮唑蓝(MTT)法测定细胞存活率,用流式细胞术检测细胞凋亡率。结果成功构建了mdr1启动子控制的CD-TK双自杀基因真核表达载体,脂质体成功转染细胞后,经C418筛选获得稳定转染细胞株。PCR结果显示,CD、TK基因均稳定存在于K562、K562／A02细胞中,RT-PCR结果显示K562／A02-CD-TK细胞有CD、TK基因特异性条带表达,而K562-CD-TK细胞无特异性条带。加入GCV、5-FC后,与K562-CD-TK细胞相比,K562／A02-CD-TK细胞存活率明显降低(在60μg／ml(GCV和600μg／ml 5-FC联合作用后两种细胞存活率分别为85．04％和41．13％)(P<0．05),凋亡率明显升高(同样浓度下分别为2．93％,10．27％)。结论mdr1启动子驱动的CD-TK双自杀基因系统可以靶向杀伤多药耐药的白血病细胞。
Objective To construct eukaryotic expression vector of double suicide gene of thymidine kinase-cytosine deaminase (CD-TK) controlled by the multidrug resistance gene (mdr1) promoter and study its targeted killing on K562 / A02 cells effect. Methods The pcDNA3-mdr1 P-CD-TK eukaryotic expression vector was constructed by molecular cloning technique and transfected into K562 and K562 / A02 cells by lipofectamine. Stably transfected cell lines were obtained by G418 screening. The transfection and expression of TK and CD genes were identified by PCR and RT-PCR, respectively. The cells were cultured with different concentrations of ganciclovir (GCV) and 5-fluorocytosine (5-FC) Cell viability was determined by MTT assay and apoptosis rate was determined by flow cytometry. Results The CD-TK double suicide gene eukaryotic expression vector controlled by the mdr1 promoter was successfully constructed. After the cells were successfully transfected with the liposome, stable transfected cell lines were obtained by C418 screening. The results of PCR showed that the CD and TK genes were stably expressed in K562 and K562 / A02 cells. The results of RT-PCR showed that the expression of CD and TK genes in K562 / A02-CD- Cell-specific bands. After addition of GCV and 5-FC, the survival rate of K562 / A02-CD-TK cells was significantly lower than that of K562-CD-TK cells (after 60μg / ml GCV and 600μg / ml 5-FC The survival rates were 85.04% and 41.13%, respectively (P <0.05), and the apoptosis rate was significantly higher (2.93% and 10.27%, respectively) at the same concentration.Conclusion The mdr1 promoter CD-TK double suicide gene system can target multi-drug-resistant leukemia cells.