A BICISTRONIC RETROVIRAL VECTOR CONTAINING MGMT AND MDR1 DRUG RRSISTANCE GENES TRANSFER INTO HUMAN UMBILICAL CORD BLOOD CD34 + CELLS TO IMPROVE COMBINATION CHEMOTHERAPY TOLERANCE
WANG Ji shi SUN DengJun LIN GuoWei FEI Jian (Department of Hematology of Affiliated Hospital of Guiyang medical college 550004; Affiliated Huashan Hospital of Fudan University, Shanghai 200040; Shanghai Institute of Cell Biology, Academy of Sciences, Shanghai 200031)
To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase ( MGMT) and multidrug resistance gene (MDR1) increase resistance to l,3-Bis(2-Chloroethyl)-1-Nitrosourea (BC-NU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric perforation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging 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 transfectced repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34 + cells was approximately 92 % and recover rate was 75 %, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 × 105 cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4. 5 to 7.8 -fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.
【Fund】： 贵州省科委基金;; 上海市博士后基金
【CateGory Index】： R73-3;
【CateGory Index】： R73-3;