Experimental Study On Immune Tolerance Of Porcine Liver Transplantation Induced By Pre-infusion Of Donor Apoptotic Lymphocyte

BackgroundOrthotopic liver transplantation(OLT)is a routine and most effective clinical procedure for patients with end-stage liver failure.However,despite standardization of surgical techniques and continuous refinements in anti-rejection therapies,long-term results of transplantation have not significantly improved.The rejection of posttransplantation is the main reason of the loss of graft and patients.While successfully preventing acute rejection episodes,current immunosuppressive treatments are unable to control chronic rejection,which is the primary cause of long-time graft loss.In addition,they cause a global immunodeficiency predisposing to severe infections and malignancies.Therefore,induction specific immunologic tolerance is the ideal method to solve problem about rejection.Contrast to immunosuppressive treatments,induction specific immunologic tolerance is superiority,for avoiding rejection fundamentally and not affecting normal function of anti-infection and immunologic surveillance.Meanwhile,induction specific immunologic tolerance is the ideal method to solving problem about rejection of xenotransplantation,because of immunosuppression of xenotransplantation need long-term,high-level,nonspecific immunosuppressive agent to maintain.So, inducing immunotolerance,handling graft rejection and preserving graft's funtion is the extremely significant subject in fields of allotransplantation or xenotransplantation. Numerous regiments leading to the indefinite acceptance of vascularized grafts have been reported in rodent models.The relevance of these models for developing a clinical protocol,however,is severely limited,as most of these strategies fail to achieve similar results when attempted in large animals.At present,there are lots of attempt to induce immunotolerance,but all fail to get outcome.Meanwhile the mechanisms of immunotolerance is not clear,so there have not any clinical protocol to apply.Therefore,transplantation tolerance defined as survival of allograft in absence any immunosuppression remains a major goal to achieve for tansplant physicians and immunologists.Apoptotic cell death can be found in many physiological and pathological processes.More and more recent researches demonstrated that the apoptotic cells can actively regulate immunity function of organisms and induce immune tolerance by means of regulating cell immunity and humoral immunity.The apoptotic cells can induce immune tolerance because they can inhibit the maturation of immature Dendric Cells which couldn't provide enough co-stimulation signals to activate T cells,and because phagocytes produce immunosuppressive factors after phagocytosing apoptotic cells.In our experiments,we established a model of liver/heart transplantation in the rat and induced immunotolerance by the donor apoptotic cells successfully.We have proposed a new viewpoint:Liver is the main site for apoptotic cells phagocytosed,and apoptotic cells have ability to induce local immunotolerance.Under this local immunotolerance circumstance,after the liver antigen-presenting cells phagocytosis of apoptotic cells,they provide immunosuppressive signals to T cells,which inducing and maintaining immune tolerance to allografts.Now,we using miniature porcine's liver transplantation as model to validate and explore our theory at the same time.Partâ… :A miniature porcine orthotopic liver transplantation model without veno-venous bypassObjective To study the key surgical technique of orthotopic liver transplantation(OLT) in Chinese inbred miniature porcine and try to find out the way to establish of stable and practical animal model.This process can also establish the model for transplantation tolerance induction in adult animals at the same time.Methods OLT were performed without veno-venous bypass(VVB)in Chinese inbred miniature porcine.The whole process including four parts:donor animal operation, donor liver modification,recipient animal operation and perioperation management. Induced anesthesia was given to all animals and after endotracheal intubation, anesthesia was maintained by combined intravenous medicine in recipients.Donor animal was exsanguinated blood for recipients via an abdominal aorta cannula.The donor liver was protected by double perfusion from abdominal aorta and portal vein. After the recipients' liver was removed,the donor's liver was introduced orthotopicly then the donor's suprahepatic vena cava was anastomosed with the recipient's side-by-side,followed by the portal vein anastomosis.The portal vein and the suprahepatic vena cava were unclamped,allowing the new liver to be perfused with the recipient's blood and ending the anhepatic phase.Then the common hepatic artery was anastomosed,and after the release of the clamp,normal circulation of the liver was restored.Subsequently the common bile tract was anastomosed side-by-side with a stent inside—called cuff method.Results 18 OLTs were performed in Chinese inbred miniature porcine.The mean operation time was(179.6±14.3)min,and the mean anhepatic phase time (27.3±3.4)min.The mean lose of blood was 422ml,and mean blood transfusion was 444ml.No one died during the operation,1 case was died on the day that the operation was undergoing.In case of lung edema and 1 case died the next day post operation in case of bile fistula.The one-week survival rate was 88.9%for the whole group.Dramatic hemodynamic and metabolism changes were identified during anhepatic phase,such as MAP and CVP decrease significant,hyperkalemia and acidosis were found.Effective management should be taken to reverse this situation.Conclusion(1)During donor liver modification,the diaphragm was reserved about 3mm around suprahepatic vena cava and was sutured by non-trauma seam in order to avoid blood leak and keep enough tenacity after anastomosis to prevent circulation dysfunction of vena cava.(2)Bile tract reconstruction was done by modified cuff technique which can make the operation simple,decreasing the complication of bile fistula and shorting operation time.(3)Decreasing the operation time of anhepatic phase is the key point in OLTs without VVB.It is necessary to improve the technique of the anastomosis of the vessel.(4)keeping the hemodynamics stability of anhepatic phase was very important for accomplishing the operation.The whole fluid injected intravenous in the operation shoud be controlled and the injection speed should be adjust according to the changes of the hemodynamics of the different phase in the OLT without VVB.(5)The establishment of animal model of OLT in porcine is not only an useful clinical training for surgeon but also a good preparation for clinical OLT without VVB.Partâ…¡:Experimental study on tolerance induction of porcine liver transplantation by pre-infusion of lymphocyte from in vitro ⁶⁰Coγ-treated donor porcineObjective To investigate the influence of preoperative transfusion of lymphocyte from in vitro ⁶⁰Coγ-treated donor porcine on allograft survival of orthotopic liver transplantation,and develop new protocols for transplantation tolerance induction in adult animals under no common immunosuppression.Methods OLT were performed without veno-venous bypass(VVB)in Chinese inbred miniature porcine.The BanNa porcine were used as donors and the Tibet porcine as recipients.The recipient porcine were administrated intravenously with donor apoptotic lymphocyte(5×10⁸)7 days before liver transplantation,or not treated.we observed the survival time.The two teams blood alanine transaminase(ALT),aspartate aminotransferase(AST),total bilirubin(TBil),total albumin(TB),albumin(ALB),total bilirubin(TBil)and liver pathological changes were observed after the transplantation.Apoptotic percentage of lymphocyte from in vitro ⁶⁰Coγ-treated was measured by flow cytometry using Annexin V/PI double staining method.Results(1)⁶⁰Coγ-treated in vitro induced lymphocyte apoptosis(48.70±6.17%)while the non-treated groups did not(0.14±0.09%)(P＜0.01).(2)Liver grafts had a median survival time(MST)of 7 days.The group infusion with donor in vitro ⁶⁰Coγ-treated lymphocyte had the same graft survival as nontreated group(MST=7days).(3)Liver grafts biopsy of two group show the same change after operation,there are pathological mild or serious acute rejection on 3th day after operation,and serious acute rejection on 6th day.(4)ALT,AST and TBil in two groups were increased significantly accompanied by decreased of TB,ALB.But the contrast of two group is nonsignificance(P＞0.05).(5)The increasing of CD4⁺T,CD8⁺T and CD4⁺CD25⁺Tr were just like the change of lymphocyte.But the contrast of two group is nonsignificance(P＞0.05).Conclusions:⁶⁰C0γinduces porcine lymphocyte apoptosis in vitro.Immunological tolerance cannot be induced by transfusion of preoperative donor ⁶⁰Coγ-treated lymphocyte in porcine liver allograft.