Effect Of Mesenchymal Stem Cells Transfected With Recombinant Adenovirus-mediated HGF Gene On The Survival Rate Of Grain Fat Grafts

Objective:1.To study the biological and homing characteristics of mesenchymal stem cells.2.To discuss the transfer efficiency of recombinant adenovirus-mediated green fluorescent protein(Ad-GFP)to MSCs and HGF expression in MSCs carrying human hepatocyte growth factor gene(Ad-HGF).3.To investigate the effect of MSCs transfected with HGF gene on the angiogenesis and the survival rate of free grain fat grafts.Methods:1.The MSCs of the Wistar rats were isolated with density gradient centrifugation,observed cell generation cycle by flow cytometry and induced to differentiate into adipose cells.The 3rd passage MSCs were transfected with AdGFP, 25 rats were randomly divided into 3 groups;delayed fluid resuscitation group(group A,10 rats),immediately fluid resuscitation group(group B,10 rats)and sham-scalded group(group C,5 rats).Rats from group A and B were established third degree scald model involving 30%total surface area(TBSA)on back,and sham-scalded model was established in rats of group C.MSCs,transfected with AdGFP, were grafted of rats by femoral vein of rats.Liver,intestinal,skin,kidney were obtained on 1d and 7d after implantation respectively,then made frozen sections. Migration and survival of MSCs were observed by fluorescence microscopy.2.The 3rd passage MSCs were transfected with Ad-GFP(the recombinant adenovirus carrying green fluorescence protein gene)at the different MOI(50,100,150, 200).At 48h post-transfection,the transfer efficiency was evaluated by flow cytometry and the best MOI would be found out.MSCs were transfected with Ad-HGF at the best MOI.HGF expression in MSCs culture medium was evaluated by ELISA.3.MSCs of male Wistar rats were collected and cultured in vitro,and transfected with Ad-GFP or Ad-HGF.Another 150 rats were randomly divided into five groups: control group(group A,30rats),MSCs group(group B,30 rats),Ad-HGF group (group C,30rats),MSCs transplanted with Ad-GFP group(groupD,30rats)and MSCs transplanted with Ad-HGF group(groupE,30 rats).Then free grain fat tissues from inguinal region,mixed with MSCs,Ad-HGF,MSCs transplanted with Ad-GFP and MSCs transplanted with Ad-HGF respectively,were transplanted into subcutaneous layer on rats back,but control group was only mixed with DMEM-LG.fat grafts were obtained on 3,5,7,14 days and 1 and 2 month after implantation,The expression of HGF and CD34 in fat grafts were detected by immunohistochemistry.The volume of fat graft were measured by messcylinder.Results:1.MSCs could adhere to the wall and appeared polymorphic.After 5th passage culture,(1～2)×10¹¹cells were obtained,Cell cycle studies showed the percentage of stem cells on stage G₀/G₁ and S+G₂+M were79.3%and 21.7% respectively.The MSCs could be induced into adipose cells.At 24h post-transplantation,the green fluorescence -positive tissue were found abundantly in the injured area of scald wounds and intestinal mucous membrane in delayed fluid resuscitation group,whereas rare in kidney and lung.Compared with immediately fluid resuscitation group and sham-scalded group,the expression of GFP in intestinal mucous membrane was higher in delayed fluid resuscitation group(P＜0.05).In addition,the expression of GFP in scald wound tissue of delayed and immediately fluid resuscitation group were higher than that of sham scalded group(P＜0.05).2.At 48h post- transfection,the transfer efficiency of Ad-GFP to MSCs reached 86.4%(the highest value)at 100 MOI,the expression of HGF in MSCs culture medium reached to the highest level after transfected with Ad-HGF at 48h,and decreased gradually following the transfection time.3 In group E,the expression of HGF was much positive at 3d post-transplantation,and decreased gradually following the transplantation time.Compared with other 4 groups,at 3,5,7,14 day posttransplantation, the expression of HGF in fat grafts of E group showed statistics significance(p＜0.05).Microvessel quantity in group E was much more than that in other groups(P＜0.05)at 5,7,14,28,60 day post- transplantation.The percent of survival volume of fat graft in E group was significantly higher than that in other groups(P＜0.05)at 28,60 day post- transplantation.Conclusion:1.MSCs would be induced into adipose cells;Transfection of target DNA into MSCs probably might not change their homing characteristics.2.The recombinant adenovirus carrying human hepatocyte growth factor gene(Ad-HGF) would transfect MSCs.The transfected cells could efficiently express the target gene. 3.MSCs transfected with Ad-HGF would express HGF,promote angiogenesis in grain fat grafts,and increase the survival rate of fat grafts.