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目的 探讨转染醛脱氢酶基因 (ALDH3)和多药耐药基因 (mdr 1)的人脐血CD3 4+ 细胞能否同时增强对活性环磷酰胺 (4 HC)和mdr 1基因靶药的抗性。方法 构建含ALDH3和mdr 1双耐药基因的逆转录病毒表达质粒G1Na ALDH3 IRES MDR1,经LipofectAMINE介导转染包装细胞 ,采用含 4 HC和长春新碱 (VCR)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 ,将含ALDH3和mdr 1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经免疫磁珠分离系统 (MACS)纯化后的人脐血CD3 4+ 细胞 ,用PCR、RT PCR、Southernblot、Northernblot、FACS和MTT等方法检测外源ALDH3与mdr 1基因在CD3 4+ 细胞中的转移和表达。结果 MACS分离纯化后的人脐血CD3 4+ 细胞纯度平均达 91% ,回收率为 72 % ,含双耐药基因重组病毒的上清最高滴度为 6 .5× 10 5CFU/ml,应用集落计数、PCR和FACS方法测定基因转导效率分别为 18.0 %、2 0 .0 %和16 7% ,未检测到辅助病毒存在 ,有P170功能的细胞占 16 0 % ,经双耐药基因修饰的脐血CD3 4+ 细胞对 4 HC的IC50 较对照组提高 3.5倍 ,对VCR和柔红霉素的IC50 较未转染细胞分别高 6 .8和 5 .5倍。结论 逆转录病毒载体介导双耐药基因转导脐血造血干 /祖细胞获高效共表
Objective To investigate whether human umbilical cord blood CD34 + cells transfected with aldehyde dehydrogenase gene (ALDH3) and multidrug resistance gene (mdr1) can simultaneously enhance the cytotoxicity of actived cyclophosphamide (4 HC) and mdr 1 target drugs Resistant. Methods The retroviral expression plasmid, G1Na ALDH3 IRES MDR1, containing dual drug resistance genes of ALDH3 and mdr1 was constructed and transfected into packaging cells by LipofectAMINE. The recombinant plasmid was cloned by medium containing 4 HC and vincristine (VCR) The virus supernatant was subjected to pingpong interaction in unidirectional GP + E86 and double-tropic PA317 packaging cells. The supernatants containing ALDH3 and mdr1 double-drug resistant recombinant viruses were isolated by immunomagnetic beads after repeated infection with cytokine stimulation Systemic (MACS) purified human umbilical cord blood CD3 4+ cells were used to detect the metastasis and expression of exogenous ALDH3 and mdr1 gene in CD3 4+ cells by PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT. Results The purity of human cord blood CD3 4+ cells after MACS isolation and purification was 91% on average, with a recovery of 72%. The highest titer of supernatant with double drug resistance gene recombinant virus was 6.5 × 10 5 CFU / ml. Counting, PCR and FACS analysis of gene transduction efficiency were 18.0%, 20.0% and 16.7%, did not detect the presence of helper virus, cells with P170 function accounted for 16%, by double resistance gene modified The IC50 of cord blood CD3 4+ cells to 4 HC was 3.5 times higher than that of the control group, and the IC50 of VCR and daunorubicin was 6.8 and 5.5 times higher than that of the untransfected cells respectively. Conclusion Retroviral vector-mediated dual drug resistance gene transduction of cord blood hematopoietic stem / progenitor cells were co-efficient