Establishment Of Transgenic Cell Strains Expressing HLIF And HOSM Gene And Their Roles In Proliferation Of Umbilical Cord Blood CD34～+ HSPC In Vitro

Objective:To construct human embryo fibroblasts cell strain stably expressing human leukemia inhibitory factor(hLIF) or human oncostatin M(hOSM) gene which are supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34~+ HSPC in vitro.Methods:Recombinant eukaryotic expression plasmids pcDNA3.0-hLIF and pcDNA3.1(-) -hOSM constructed in our laboratory were confirmed by PCR (polymerase chain reaction) and DNA sequence determination . The two recombinant plasmids were transfected into human embryo lung fibroblasts cell line WI-38 by Lipofectamine reagent respectively. Cell clones which stably expressed hLIF or hOSM were obtained in the medium which contained G418 after 3-4 weeks. Try to construct recombinant retrovirus vectors of hLIF and hOSM,human embryo kidney fibroblasts modified by hLIF or hOSM gene via retrovirus were acquired in the medium which contained zeocin. Objecive genes were detected by RT-PCR,ELISA and biological methods on mRNA and protein level. Transgenic human embryo fibroblasts cell strains were treated by MMC and then used as feeder layers for umbilical cord blood CD34~+ HSPC separated by magnetic-activated cell sorting (MACS). Cell numbers and cell surface markers such as CD34, CD49d, CXCR4, CD54, CD31, CD62L were detected in order to observe the effects of hLIF and hOSM modified WI-38 on the proliferation and homing potential of cord blood CD34~+ HSPC in vitro .Vectors without objective genes were as control in this experiment.Results:The results of PCR and DNA sequence proved that the recombinant expression plasmids pcDNA3.0-hLIF and pcDNA3.1(-)-hOSM are correct. The recombinant retrovirus vector pEGZ/MCS-HA-hLIF and pEGZ/MCS-HA-hOSM were confirmed by PCR and endonucleases digestion and the full cDNA sequence of hLIF and hOSM are agree with Genbank. Result of RT-PCR showed there were expected bands of about 609bp and 750bp, which demonstrated hLIF and hOSM gene could be transcripted in WI-38 cells. Similarly, hLIF and hOSM gene could be expressed in human embryo kidney fibroblasts cell line 293T transfected via retrovirus. The concentration of hLIF in the supernatant of WI-38-hLIF was about 11.105 pg/ml, 293T-hLIF 110.134 pg/ml. The concerntration of hOSM in the supernatant of WI-38-hOSM was about 49.547 pg/ml, 293T-hOSM 159.987 pg/ml. The supernatants had natural biological activities, for instance they could inhibit the growth of HL60 and A375 respectively. The purity of CD34~+ cells from umbilical cord blood separated by MACS could reach to 90% and the yielded about 45%. The rates of adhension molecules'expression on the surface of CD34~+cells were CD31 89.33%, CD62L 23.60%, CD49d 85.4%, CD54 74.9%, CXCR4 76.6%. After culturing for 7 days,CD34~+ cells were more in the group containing hOSM than without hOSM; more in the group containing both cytokines and stromal cells than in the single cytokines group; and also more in the group of transgenic stromal cells. Moreover, CXCR4 expression was higher in the group containing hOSM than without hOSM culture medium.Conclusion:Human embryo fibroblasts cell strains stably expressing hLIF or hOSM gene were obtained and the supernatants had their own biological activities. Recombinant retrovirus vector pEGZ/MCS-HA-hLIF and pEGZ/MCS-HA- hOSM were successfully constructed. CD34~+HSPCs were successfully separated from umbilical cord blood by the way of MACS and the purity could reach to 90%. Experimental results indicated that transgenic human embryo fibroblasts modified by hLIF and hOSM could provide a better microenvironment for the growth and proliferation of umbilical cord blood CD34~+ HSPC in vitro which contributes to maintain its multipotency and undifferentiation state.