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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 investigate whether the human umbilical cord blood CD34 ~ + cells transfected with aldehyde dehydrogenase gene (ALDH1) and multidrug resistance gene (MDR1) can simultaneously enhance the resistance to cyclophosphamide (4-HC) and MDR1 gene targets The retroviral expression plasmid G1Na-ALDH1-IRES-MDR1 containing both the dual drug resistance genes of ALDH1 and MDR1 was constructed and transfected into GP + E86 and PA317 cells by Lipofect-AMINE. Vincristine (VCR ) And 4-HC were selected and cloned. The recombinant virus supernatants were collected and subjected to ping-pong cross-infection with unidirectional GP + E86 and double-type PA317 packaging cells to obtain PA317 recombinant virus-producing cells with the highest titer of 5.6 × 10 ~ 5CFU / ml). The supernatant of ALDH1 and MDR1 double-drug resistant recombinant viruses were infected by CD34 + cells repeatedly and stimulated by cytokines. The expression of CD34 ~ + was detected by RT-PCR, Southern blot, Northern blot, MTT and other methods to detect exogenous ALDH1 and MDR1 gene in CD34 ~ + cells in the transfer and expression. The results showed that the retroviral vector-mediated double-drug resistance gene has been integrated into the transfected target cell genome and obtained effective expression, while transmitting different resistant phenotypes. The double-drug resistant gene-modified cord blood CD34 ~ + cells simultaneously produced resistance to 4-HC and VCR drugs with IC50 value of 4 times and 7.2 times higher than that of untransfected cells respectively. In this study, The clinical research of treatment has laid the experimental foundation.