The Experimental Research On Purification, Expansion And Transduction Of Multidrug Resistance Gene (MDR1) Of Peripheral Hematopoietic Stem Cells

Objective: Our research was designed to investigate the appropriate combination of hematopoietic growth factors (HGF) and increase MDRl transfer efficiency of retrovirus-mediated transduction into peripheral blood stem cells (PBSC).Materials and methods: From May 2001 to May 2002, we enrolled 22 patients with malignant tumors eligible for autologous peripheral blood stem cell transplantation in Tianjin cancer hospital. 11 patients were male, and the others were female. The median age was 38.8 years. PBSC were harvested after mobilization with CBP + Vp16 + G-CSF + GM-CSF. CD34+ cells were isolated by using a high-gradient magnetic cell sorting system (MACS), and expanded with different HGF combination in a liquid culture system. There were six groups: (1)control, without any HGF; (2)SCF + IL-3 + GM-CSF + TPO; (3)FL + GM-CSF + IL-4; (4)SCF + IL-3 + FL + TPO; (5)SCF + IL-3 + IL-6 + FL + GM-CSF; (6)SCF + IL-3 + FL + TPO + EPO. We characterized the expanded cells by total cells counting, flow cytometry and colony forming units. MDRl was transferred into PBSC by retrovirus in the presence of HGF and RetroNectin. We examined the transfection and expression of MDRl by Rhodamine 123 efflux assay, RT-PCR, competitivePCR, colony forming units culture after exposure to Taxol and MTT colorimetric assay.Results: The proportion of CD34+ cells in mobilized PB was l.3± 0.28%. Overall yield was 62.39±5.21% and overall purity was 90.3±4.35%. The proliferation and clonogenic potential of CD34+ cells were significantly higher in the presence of SCF + 1L-3 + FL + TPO + EPO, and the percentage of CD34+ cells in total cells were highest. MDR1 was tranducted into PBSC by retrovirus in the presence of HGF and RetroNectin. Gene tansfer efficiency was over 20%, and the resistance to Taxel and Vincristime was higher.Conclusion: The gene transfer efficiency was over 20% in the presence of SCF + IL-3 + FL + TPO + EPO and RetroNectin. The efficiency of this gene transfer system provides a foundation for ameliorating combination chemotherapy toxicity in clinical trial.