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目的 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 +细胞能否同吮增强对活性环磷酰胺 ( 4 HC)和P gp转运泵靶药的抗性。方法 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LipofectAMINE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与双嗜型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耐药基因重组病毒上清在细胞生长因子刺激下重复感染人脐血CD34 +细胞。结果 经PCR ,RT PCR ,Southernblot,Northernblot,FACS和MTT方法检测显示外源ALDH1与MDR1基因已经整合入转染靶细胞中基因组并获得有效表达 ,同时传递不同的耐药表型。经耐药基因修饰的脐血CD34 +细胞对 4 HC和P gp转运泵靶药同时产生抗性 ,其IC50值分别比未转染细胞高 4倍 ( 4 HC) ,5 5倍 (柔红霉素 DNR)和 7 2倍 (VCR)。结论 双功能逆转录病毒载体介导两种不同耐药基因转染人脐血CD34 +细胞能增强联合化疗抗性 ,本基因转移系统的建立为开展肿瘤基因治疗的临床研究奠定了实验基础。
Objective To investigate whether human cord blood CD34 + cells transfected with aldehyde dehydrogenase gene (ALDH1) and multidrug resistance gene (MDR1) can enhance the activity of cyclophosphamide (4 HC) and P gp transport pump targets. Resistance. METHODS: The retroviral expression plasmid G1Na ALDH1 IRES MDR1 containing both ALDH1 and MDR1 resistance genes was constructed and then transfected into GP + E86 and PA317 packaging cells by LipofectAMINE and incubated with vincristine (VCR) and 4 HC. After the clonal selection, the recombinant virus supernatants were collected and ping-pong cross-infection was performed between the one-way and double-tropic packaging cells to obtain PA317 recombinant virus producing cells (the highest titer was 5 6×10 5 CFU ml), which would contain ALDH1 and MDR1 resistance. The drug gene recombinant virus supernatant was repeatedly infected with human umbilical cord blood CD34 + cells stimulated by a cell growth factor. Results PCR, RT PCR, Southern blot, Northern blot, FACS and MTT assays showed that exogenous ALDH1 and MDR1 genes were integrated into the genome of the transfected target cells and effectively expressed. At the same time, different resistance phenotypes were transmitted. CD45 + cells modified by drug-resistant genes are resistant to 4 HC and P gp transport pump targets, and their IC50 values are 4 times higher (4 HC) and 55 times higher (non-transgenic) DNR) and 7-fold (VCR). Conclusion Bi-functional retroviral vectors mediating the transfection of human umbilical cord blood CD34 + cells with two different drug resistance genes can enhance the resistance to chemotherapy. The establishment of this gene transfer system lays the foundation for the clinical research of tumor gene therapy.