逆病毒载体介导双耐药基因在脐血CD34^+细胞中的表达和抗性研究  

Study on Expression and Resistance of the Double Drug Resistance Genes Transduced into Human Umbilical Cord Blood CD34^+ Cells Mediated by Bicistronic Retroviral Vector

作　　者：王季石[1] 夏学鸣[2] 陈子兴[2] 卢大儒[3] 薛京伦[3] 阮长耿[2] 

机构地区：[1]贵阳医学院附院血液科 [2]江苏省血液研究所苏州医学院附一院血液科,苏州215006 [3]复旦大学遗传学研究所,上海200433

出　　处：《实验生物学报》2000年第4期341-348,共8页Acta Biologiae Experimentalis Sinica

基　　金：国家自然科学基金;贵州省科委基金

摘　　要：为探讨转染醛脱氢酶基因(ALDH1)和多药耐药基因(MDR1)的人脐血CD34^+细胞能否同时增强对活性环磷酰胺(4-HC)和MDR1基因靶药的抗性,构建了同时含ALDH1和MDR1双耐药基因的逆转录病毒表达质粒G1Na-ALDH1-IRES-MDR1,经Lipofect-AMINE介导转染GP+E86和PA317包装细胞,采用含长春新碱(VCR)和4-HC的培养基克隆选择后收集重组病毒上清于单向型GP+E86与双嗜型PA317包装细胞行乒乓交互感染,获得PA317重组病毒生产细胞(最高滴度达5.6×10~5CFU/ml),将含ALDH1和MDR1双耐药基因重组病毒的上清在细胞生长因子刺激下重复感染人脐血CD34^+细胞,用PCR、RT-PCR、Southern blot、Northern blot、FACS和MTT等方法检测外源ALDH1与MDR1基因在CD34^+细胞中的转移和表达。结果显示:逆转录病毒载体介导的双耐药基因已经整合入转染靶细胞基因组并获得有效表达,同时传递不同的耐药表型。经双耐药基因修饰的脐血CD34^+细胞对4-HC和VCR药物同时产生抗性,其IC_(50)值分别比未转染细胞高4倍和7.2倍,本研究为开展肿瘤基因治疗的临床研究奠定了实验基础。To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and mul-tidrug resistance gene (MDRl) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed . The vector was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone? we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5. 6×105 CFU/ml. Cord blood CD34+ cells were trans-fectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDRlcDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+cells and expressed efficiently. The transgenes recipient cells confered 4-to 7. 2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontrans-duced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.

关 键 词：耐药基因 逆病毒载体介导 脐血CD34^+细胞 表达 

分 类 号：R346[医药卫生—基础医学]