Contrastive Study About The Effect And Gene Expression Of Three Gene Vector Transfecting Human Umbilical Cord Wharton’s Jelly-derived Mesenchymal Stem Cells

Background Gene therapy is emerging as a potential important treatment option ofcardiovascular diseases. However, progress in the field of gene therapy for cardiovasculardisease has been modest; one of the key reasons for this limited progress is the lack of genedelivery systems for localizing gene therapy to specific sites to optimize transgeneexpression and efficacy.Mesenchymal stem cell derived from Human umbilical cord Wharton’s jelly has moreadvantages which make it becomes a potential seed cells of clinical treatment.Transplanting hUCMSCs with specific gene into vivo, the stem cells automatically migrate tothe diseased areas, then self-renewal and self-differentiation in the areas, meanwhile, theintroduction of transgenes will providing long-term stable expression. The transplanting notonly solves the lack of specific cells, but also provides a new molecular biological treatmentmethod of gene transporting, gene targeting and long-term gene expression. It will be abreakthrough for treatment of cardiovascular disease.Gene transfection requires certain vectors to transduce exogenous gene into the biologicalcell, the biggest obstacle is the safety and efficiency of the vectors, so the study of vectorsto improve gene therapy technology is also necessary.Objective1Isolate MSCs from human umbilical cord Wharton’s jelly, observe the cell morphology,identify the cell phenotype, prepare seed stem cells for building genetically engineeredcells.2Comparative study about the transfection efficiency and gene expression of Adenovirusvector mediated EGFP, Recombinant Adeno-Associated Virus vector mediated EGFP and Lentivirus vector mediated EGFP in transfecting hUCMSC.Methods1A fresh and asepsis umbilical cord was obtained from cesarean section. Subsequently,after removal of the umbilical arteries and vein, separate and collect the mesenchyme fromtunica externa.Then wharton’s jelly of umbilical cord was cut into pieces and cultured byDMEM/F12medium. Transfer of culture were undergone when these adherent cells fusionwere about80%. The second generation of cells was detected cytoactive and phenotypesincluding CD44, CD90, CD105, CD73, CD34, CD45, HLA-DR, HLA-ABC with flow cytometryand identified by differentiating into osteogenic and adipogenic tissues.2. The third generation of hUCMSC were transfected by adenovirus vectors, lentivirusvectors and adeno-associated viruse vectors, both of the three were mediated wih EGFP.Observed EGFP in cells with fluorescence microscope in day1、3、7、14、21，and checkedout EGFP gene and protein level in collected cells by fluorescent quantitation PCR andWestern Blotting in day7、14and21.Results1hUCMSCs presenting with long spindle shape were observed in1-2weeks or so. In the3rdweek, transfer of culture was done in new culture flasks with density of1×106cells/75cm2every3-5days, when cells fusion were over80%. Each generation were homoemorphy inmicroscope and proliferation capacity had not dropped in the ultimate generation of P20.Results of phenotypes in P2with flow cytometric testing shows that matrix cells antigensincluding CD73、CD90、CD44、CD105and HLA-ABC were positive and expression ofhematopoietic stem cell antigens including CD34、CD45and HLA-DR were negative.Immunohistochemistry showed that the MSCs strongly expressed alkaline phosphatase andVon Kossa positive.21) The optimal MOIs of Adenovirus vector mediated EGFP, RecombinantAdeno-Associated Virus vector mediated EGFP and Lentivirus vector mediated EGFP in transfecting hUCMSC are different, they are1×104,1×105,10. When at the optimal MOI,the transfection efficiency of Ad5F35-mCMV-EGFP was higher than rAAV2-EGFP andLV-EGFP, the difference was statistically significant（p<0.05）.2) When at the optimal MOI,the EGFP gene and protein expression level of Ad5F35-mCMV-EGFP was specially higherthan rAAV2-EGFP and LV-EGFP, the difference was statistically significant（p<0.05）.3) AfterhUCMSCs were transfected by Ad5F35-mCMV-EGFP, rAAV2-EGFP and LV-EGFP, theexpression of EGFP gene and EGFP protein presented different rules. AfterAd5F35-mCMV-EGFP transfected hUCMSC6hours, the EGFP visible expression could berecorded, but the visible EGFP expression of rAAV2-EGFP and LV-EGFP were3~5days aftertransfecting, later than the Ad-EGFP team. EGFP gene could continuous and stable expressin the rAAV2-EGFP team and LV-EGFP team, but couldn’t in the Ad-EGFP team. Observed5weeks after transfecting.4) Recombinant Adeno-Associated Virus vector mediated EGFPsuccessfully transfected hUCMSCs, it would suggest that different type demonstratesdifferent transfection characteristics.Conclusion1. hUCMSCs were successfully obtained from human umbilical cord Wharton’s jelly bytissue adherent method, they meet the basic criteria of MSCs announced by ISCT in2005.2. hUCMSCs were successfully transfected by adenovirus vectors, lentivirus vectors andadeno-associated viruse vectors, both of the three were mediated wih EGFP. And selectedthe LV-EGFP as the optimal transfection vector, and gained a new understanding of therAAV.3. Lentivirus vectors mediated with EGFP successfully transfected hUCMSCs, the explorationof the optimal MOI and transfection condition lays a foundation for constructing geneticengineering cells next.