The Protective Effect And Its Mechanism Research Of MSCs In Liver Graft Of A Mouse Non-Heart-Beating Liver Transplantation Model

Part ?:Establishment of a Standardized Mouse Non-Heart-Beating Liver Transplantation Model[Objectives]Liver transplantation is the only cure of end-stage-liver disease.Organ shortage is a severe problem for development of liver transplantation.Donor of Cardiac Death(DCD)donation is an important strategy to fill the gap between donors and recipients.However,because of unavoidable ischemic/reperfusion injury(IRI),DCD donation may cause a series of problems such as primary graft dysfuction,early graft dysfunction,delayed graft dysfunction and biliary tract complications.How to alleviate IRI injury to improve the quality of DCD grafts become a hotspot recently.Mouse has inimitable advantages in researches of transplant immunology and IRI,however,no preferable mouse DCD liver transplantation models are available for researchers.Thus,a standardized mouse Non-Heart-Beating liver transplantation model need to be established urgently.[Methods]We have established a standardized mouse Non-Heart-Beating liver transplantation model with hepatic artery reconstruction.Utilizing this model,we researched the survival rate and pathological changes of grafts with varing warm ischemic time after transplantation to ascertain the best conditions of this model.[Results]Donor liver graft was harvested and transplanted at Omin,5min,10min and 20min after cardiac arrest,and the 7-day survival rate was 100%,75%,37.5%and 12.5%,respectively.At 6h post-transplant,serum ALT/AST significantly elevated with the extension of donor cardiac arrest time.Histology examination showed graft hepatocytes changed from mild edema to spotty necrosis to patchy necrosis.Besides,cytokines intro-graft such as IL1?,IL1?,TNF?,IFNy,IL6,IL10 and Kupffer cells surface marker F4/80 were generally down-regulated post transplantation.[Conclusions]Along with the extension of donor cardiac arrest time,IRI of donated liver graft exacerbated with an obvious descending in quality.After liver transplantation,intrahepatic immune cells showed a status of impaired immune response,and Kupffer cells lost due to severe damage,which together resulted in significant decline of recipients' survival rate.Controling warm ischemic time at 10min and cold ischemic time at 2h was the optimal conditions of this mouse model which would garantee a certain survival rate and a convenient observation in judging whether interventions are valid or not.Part ?:The protective effect research of MSCs in liver graft of a mouse Non-Heart-Beating liver transplantation model[Objectives]Mesenchymal Stem Cells(MSCs)are a subpopulation of multipotent cells,which possess powerful immunemodulatory functions and have been researched in inflammation,IRI,autoimmune diseases.Reciently,MSC have been utilized in liver diseases such as fulminant hepatitis,liver failure and even hepatocellular carcinoma.In liver transplantation,MSCs have raised more and more interest and attentions due to its inhibitory effect on immune rejection and dose reduction of immunosuppressor.However,whether MSCs can protect DCD donated liver graft still remines unclear.[Methods]Utlizing our model metioned above,we controlled cardiac arrest time at 10min,and investigated the effect of MSCs infusion on DCD donated liver graft after liver transplantation.Survival rate,liver function and pathological examination were surveyed to evaluate the protective effect of MSCs.[Results]MSCs infusion significantly elevated the 7-day survival rate from 37.5%to 100%,and decreased the ALT/AST levels and hepatic injury of liver grafts.Pathological examination showed extreme mild edema of hepatocytes in MSC infusion groups,which distinguished with severe edema and spotty necrosis in control group.Meanwhile,3 months after transplantation,the survival rate of MSC infusion group still remained 75%,liver function and histology of graft were normal.[Conclusions]MSCs infusion could protect DCD donated liver graft,alleviate liver injury and promote survival post-transplantation.Prolonged observation demenstrated the safety and effectiveness of MSCs infusion without any risks of tumorigenesis.Part ?:The mechanism research of MSCs in liver graft of a mouse Non-Heart-Beating liver transplantation model[Objectives]Warm ischemic injury is the greatest challenge in DCD donated liver graft.With the loss of effective circulatory perfusion,liver endures hypoxia status,and nonparenchymal cells are quite sensitive to hypoxia.Among them,Kupffer cells'response to pathological factors play a critical role in hepatic immune state.In above experiments,we proved Kupffer cells decreased in DCD donated grafts and MSCs could protect DCD graft injury.But the connections between MSCs and Kupffer cells are still unknown.What's more,the protective mechanism of MSCs need to be investigated.[Methods]GdCl3 was used to eliminate Kupffer cells in graft,then MSCs infusion with liver transplantation were conducted to define their connections.Metheds such as qRT-PCR,immunoassays,immunohistochemical staining and in vitro cell co-cultrue were used to reveal certain underlying mechanism of MSCs.[Results]MSCs infusion promoted expression of F4/80 on Kupffer cells membrane surface.While MSCs lost its protection on liver graft when intrahepatic Kupffer cells was eliminated by GdCl3 prior to transplantation.In MSC infusion group,expression of proinflammatonry cytokines and chemokines,infiltration of immune cells were both down-regulated significantly.In vitro co-culture experiment demonstrated MSCs could inhibit apoptosis of Kupffer cells,and NS-398,a PGE2 inhibitor could reverse MSCs'protection.Western blot analysis revealed MSCs could secrete PGE2 to inhibit TLR4 expression on Kupffer cells,activate ERK1/2,reduce Fas/FasL expression and inhibit activation of caspase3,which finally inhibit apoptosis of Kupffer cells.[Conclusions]MSCs could ruduce Kupffer cells apoptosis during IRI in a PGE2 denpendent way.TLR4-ERK1/2-Fas/FasL-caspase3 signaling pathway was involved in this protetive effect.Thus,proinflammatonry cytokines and chemokines expression,immune cells infiltration and nonparenchymal cells apoptosis were down-regulated,finally prolonged recipient survival of DCD donated liver transplantation.