A bicistronic retroviral vector to introduce drug resistance genes into human umbilical cord blood CD34+ cells to improve combination chemotherapy tolerance
10.3760/j.issn:0366-6999.2001.01.005
- VernacularTitle:双功能逆转录病毒载体介导耐药基因转染人脐血CD34+细胞能增强联合化疗抗性
- Author:
Jishi WANG
1
;
Zixing CHEN
;
Xueming XIA
;
Daru LU
;
Jinglun XUE
;
Changgeng RUAN
Author Information
1. 苏州大学附属第一医院
- Keywords:
gene therapy;
aldehyde-dehydrogenase-class1gene (ALDH-1);
multidrug resistance gene (MDR1);
retrovirus vector;
gene co-expression;
hematopoietic stem cells;
cord blood
- From:
Chinese Medical Journal
2001;114(1):25-29
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. Methods A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP+E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6×105 CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. Results Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. Conclusion The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.
