The proliferation of umbilical cord blood CD34+ HSPC in transgenic cell strains of hLIF and the experimental study of engraftment in SCID mice.

Objective:To establish the transgenic cell strains expressing recombinant adenovirus vector of hLIF gene which was supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34+ HSPC in vitro and study the SCID mice model of HSPC transplantation.Methods:The recombinant adenovirus vector of hLIF gene was transfected into human embryo kidney fibroblasts cells 293A and the human embryo lung fibroblasts cells WI-38 were infected with the recombinant adenovirus,and the objective gene was detected by RT-PCR.The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting (MACS) was detected by flow cytometry.After culturing with feeder layer cells for 7 days,the rate of proliferation was detected by flow cytometry.CD34+ HSPCs stained by CFDA SE was injected to the sublethally irradiated SCID mice.Humanized gene was detected by RT-PCR and fluorescence microscope.Results:The green fluorescence was observed by fluorescence microscope in the transgenic cell strains,and the objective gene was confirmed by RT-PCR.The purity of umbilical cord blood CD34+ HSPC separated by magnetic-activated cell sorting (MACS) could reach to (95.6±2.58)%.After culturing with feeder layer cells for 7 days,the CD34+ cells were 13.2 times in group containing hLIF than in group without hLIF.The rates of adhensire molecules’ expression on the surface of CD34+ cells were also higher in the group containing hLIF than without hLIF.Four days after transplanted in SCID mice,fluorescently-labeled humanized cells could still be observed.The existence of the humanized gene Alu was confirmed by RT-PCR.Conclusion:Recombinant adenovirus vector of hLIF gene as feeder layer cells can effectively enhance proliferation of umbilical cord blood CD34+ HSPC in vitro and delay their differentiate,what’s more,it has high transplant efficacy and haematogenesis activity.