造血干/祖细胞遗传学修饰对4-氢过氧化环磷酰胺、长春新碱和柔红霉素的联合抗性(英文)

目的:探讨经醛脱氢酶基因(ALDH-3)和多药耐药基因(MDR1)遗传学修饰的人外周血造血干/祖细胞能否同时增强活性环磷酰胺(4-HC)和MDR1基因靶药的抗性。方法:构建同时含ALDH-3和MDR1双耐药基因的逆转录病毒表达质粒G1Na-ALDH3-IRES-MDR1,经电穿孔导入PA317包装细胞,将免疫磁珠分离系统(MACS)分离纯化后的人外周血CD34~ 细胞用含有ALDH-3和MDR1双耐药基因重组病毒的上清感染,用PCR、RT-PCR、Southern blot、Nor-thern blot、ACS和MTT等方法检测外源ALDH-3与MDR1基因在CD34~ 细胞中的转移和表达。结果:用PCR与酶切分析法鉴定了双顺反子逆转录病毒载体构建的正确性,双耐药基因已整合入转染靶细胞中基因组,并获得有效表达,应用集落计数测定基因转导效率为18％。巢式PCR及补救分析均未检测到辅助病毒存在。经双耐药基因修饰的CD34~ 造血干/祖细胞对4-HC的IC_(50)较对照组提高4.5倍,对多药耐药基因靶药(长春新碱和柔红霉素)的IC_(50)较未转染细胞分别高6.6和7.8倍。结论:逆转录病毒载体介导双耐药基因转导外周血造血干/祖细胞高效共表达可降低联合化疗骨髓毒性。

Genetic modification of hematopoietic progenitor cells for combined resistance to 4-hydroperoxycyclophosphamide, vincristine, and daunorubicin

AIM: To investigate whether human peripheral blood hematopoietic progenitor cells (PBPC) modified with human aldehyde dehydrogenase class-3 gene (ALDH-3) and multidrug resistance gene 1 (MDR1) would increase chemotherapy resistance to 4-hydroperoxycyclophosph-amide (4-HC) and P-glycoprotein effluxed drugs. METHODS: A bicistronic retroviral vector G1Na-ALDH3-IRES-MDR1 cDNA was constructed and used to transfect the packaging cell lines PA317 by electropora-tion. CD34 PBPC were isolated with a high-gradient magnetic cell sorting system (MACS), and then were transfectced with supernatant of retrovirus containing human ALDH-3 and MDR1 cDNA. PCR, RT-PCR, Southern blot, Northern blot, FACS, and MTT assay were used to evaluate the transfection and expression of the transgene in target cells. RESULTS: The bicistronic retroviral vector construction was verified by PCR and restriction endonuclease analysis. Dual drug resistance genes were integrated into the genomic DNA of CD34 PBPC and expressed efficiently. The efficiency of gene transfection in CD34 PBPC was tested to be 18 % on colonies. Nested PCR and Neor rescue assayindicated that no helper virus was present in this system. Compared with the untransduced cells, transgene recipient cells confered 4.5-fold resistance to 4-HC, 6.6-fold and 7.8-fold resistance to P-glycoprotein effluxed drug, vin-cristine and daunorubicin, respectively. CONCLUSION: Efficient transduction of two different types of drug resistance genes into human peripheral blood hematopoietic progenitor cells and the co-expression may decrease cumulative myelosuppression of combination chemotherapy.