A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34+ cells to improve combination chemotherapy tolerance.

摘要: Objective To study whether human umbilical cord blood CD34+ cells transduced with aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases to 4-Hydroperoxycyclo-phosphamide (4-HC) P-glycoprotein effluxed drugs. Methods A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed used transfect the packaging cell lines GP + E86 PA317 by LipofectAMINE method, using medium containing VCR 4-HC agents for cloning selection ping-ponging supernatant infection between ecotropic producer clone amphotropic clone, we obtained high titer producing titers up 5.6 x 10(5) CFU/ml. Cord were transfected repeatedly of retrovirus ALDH-1 MDR1cDNA under stimulation hemopoietic growth factors. Results Bicistronic construction verified restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern fluorescenceactivated sorting (FACS) method 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug genes been integrated into genomic DNA expressed efficiently. The transgenes recipient confered 4-fold stronger 5.5 7.2-fold than untransduced cells. Conclusion vector-mediated transfer two different types co-expression provided an experimental foundation improving combination chemotherapy tolerance in tumor clinical trial.