mdr1启动子驱动的CD-TK双自杀基因载体对耐药K562细胞的体外靶向杀伤作用  

Study on the targeted killing effects of CD-TK double suicide genes driven by mdr1 promoter on mul-tidrug-resistant K562 cell line in vitro

作　　者：王向玲[1] 纪春岩[1] 马道新[1] 赵建强[1] 侯明[1] 余海青[1] 臧绍蕾[1] 

机构地区：[1]山东大学齐鲁医院血液科,济南250012

出　　处：《中华血液学杂志》2006年第11期757-761,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目(30471941);山东省科技发展计划项目(031050104);山东省科技发展计划项目(2002BB1CJA3)

摘　　要：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 the CD（ cytosine deaminase） and TK（ thymidine kinase） double suicide genes expressive vector driven by mdrl promoter and to investigate its target killing effects on mdrl positive leukemia cell line K562/A02 in vitro. Methods The expressive vector pcDNA3-mdrl P-CD-TK containing CD-TK double suicide genes driven by mdrl promoter was constructed and transfected into K562 or K562/A02 cells using lipofectin. Stable transfectant subline was established after geneticin（ G418 ） selection. PCR and RT-PCR were used to identify the stable transfection and expression of CD and TK gene. The stable sublines were cultured with ganciclovir（GCV） and 5-fluorocytosine （5-FC） at different concentrations for 4 days, respectively. The in vitro cytotoxic effects of pcDNA3-mdrl P-CD-TK/GCV ＋ 5-FC system were meas- ured by M3T assay. The apoptotic ratio was observed by flow cytometry. Results The pcDNA3-mdrl P-CDTK expression vectors were successfully constructed. Double suicide genes were transfected stably into the K562 cells and K562/A02 cells. CD and TK gene were expressed in K562/A02 cells, but not in K.562 cells. Compared with that of K562-CD-TK（ 85.04% ）, the survival ratio of K562/AO2-CD-TK cell was decreased to 41.13% after treatment with 60μg/ml of GCV and 600 p,g/ml of 5-FC （P 〈 O, 05 ）. The apoptotic ratios were 2.93% and 10.27% respectively at the same concentration. Condusion The CD-TK double suicide genes system driven by mdrl promoter has targeted killing effect on MDR positive leukemia cells K562/A02.

关 键 词：基因 mdrl 自杀基因 基因治疗 K562细胞 

分 类 号：R733[医药卫生—肿瘤]