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CD34+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-l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PA.317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CD34＋细胞，用PCR、RT-PCR、Southern blot、Northern blot、FACS和MTI等方法检测外源ALDH3与mdr-1基因在CD34＋细胞中的转移和表达。结果MACS分离纯化后的人脐血CD34＋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6.5×105 CFU/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％、20.0％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CD34+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/祖细胞获高效共表达。%Objective To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human aldehyde dehydrogenase class 3 (ALDH3) and multidrug resistance gene (MDR1) could increase resistance to 4-hydroxycyclophosphamide (4-HC) and P-glycoprotein effiuxed drugs. Methods A bicistronic retrornviral vector G1Na-ALDH3-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by LipofectAMINE method , using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34 +cells were enriched with a high-gradient magnetic cell sorting system(MACS), and then repeatedly transfected with rnsupernatant of retrovirus containing human ALDH3 and MDR1 cDNA under stimulation of hematopoietic growth factors. PCR , RT-PCR, Southern blot, Northern blot, FACS and MTT assay were used to evaluate the transfection and expression of the double genes. Results The purity of cord blood CD34 ＋ cells was approximately 91% and the recovery rate was 72 %. The highest titer of recombinant amphotropic retrovirus in the supernatant was up to 6.5 ×105 CFU/mI. The efficiency of gene transduction was 18% ,20% and 16.7% tested by colony formation, PCR and FACS, respectively. Rhodamine 123 effiux showed 16% transduced cells with P-gp function. No helper virus was found by beth nested PCR and rescue assay. The MTT analysis showed a 3.5 to 6.8-fold increase of resistance of transducted cells to cyclophosphamide and P-glycoprotein effiuxes drug as compared with the nontransduced cells.Conclusion The efficiency and co-expression of this dual genes transfer system provided a foundation for amelioratrning combination chemotherapy toxicity in clinical trial.
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