The Study Of Liver Transplantation Immune Tolerance Induced By Donor Hepatocytes In Rats And Its Mechanism

Part 1Induction of liver transplantation tolerance by donor-derived hepatocytes infusion:an experimental study in ratsBackground:Immunologic rejection is a key factor in the hindrance of the organ transplantation development.Immunologic rejection in liver transplantation is of considerable light when comparing with the cardiac and kidney transplantation.However,results of clinical liver transplantation have shown that the rejection and loss of human liver allograft occurs despite the use of potent immunosuppression,furthermore,immunosuppressive drugs invites side effects in the form of an increased risk of opportunistic infections and posttransplant malignancies,at the same time,this treatment also proves financially exhausting for the patients and their families in the long term.Therefore,inducing autoimmune tolerance and understanding the basis of allograft tolerance in liver transplantation is playing a new crucial role in the immunosuppressive treatment.It is apparent that the liver possesses unique tolerogenic properties,and acceptance of a liver graft can induce specific tolerance to subsequent transplants of other solid organs,which would otherwise be rejected. Accordingly,we considered that the donor hepatocytes itself play an important role in tolerance induction and maintenance.In order to assess the impact of donor-derived hepatocytes on the induction of liver transplantation immune tolerance,we worked out this animal experiment.The clinical significance of our study is mainly service living donor liver transplantation(LDLT),because it is very difficult to get donor hepatocytes in advance in cadaveric liver transplantation;but as for LDLT,we can obtain donor hepatocytes previously by laparoscopic biopsy;at the same time,we can also get the information about donor liver quality by direct-viewing.Objective:To assess the impact of donor-derived hepatocytes on the induction of liver transplantation immune tolerance using a rat model.Methods:Wistar rats and SD rats were divided randomly into three groups(n=15),ten recipients were used to do detection and other 5 recipients were used to observe live time every group.Liver transplantation was performed from Wistar rats to SD rats.Hepatocyte group:1ml donor hepatocytes(2×10~7/ml)were injected into recipients through the vena dorsalis penis,liver transplantation was performed on day 14 after injection.Bone marrow cell(BMC)group:1ml BMC(2×10~7/ml)was injected into recipients through the vena dorsalis penis,liver transplantation was performed on day 14 after injection.Control group:1ml Sodium Chloride was injected into recipients through vena dorsalis penis,liver transplantation was performed on day 14 after injection.Rejecting the death rat within 24 hours postoperatively,recipient animals for detection were sacrificed on day 7 after transplantation;survival rate on postoperative 7 days were calculated,blood and tissue from liver was sampled from animals for serological(ALT,AKP and ALB)and histological evaluation.Death time of recipients which were used to observe live time was recorded and was compared each other among three groups.Results:Compared with BMC group and control group,the donor hepatocytes significantly increased the SVR,the level of ALB and the number of FasL positive hepatocytes,prolonged the survival time,decreased the RAI and the level of ALT and AKP of recipient rats;whereas bone marrow cells failed to improve those of recipient rats.Conclusions:(1)Liver parenchymal cells significantly increased the SVR and survival time of recipients.(2)Liver transplantation immune tolerance of recipients was induced by donor hepatocytes,whose effect was surpass that of BMC.Part 2The study of the adoptive transfer and specificity of liver transplantation immune tolerance induced by donor-derived hepatocytesBackground:The findings of part 1 proved that the donor-derived hepatocytes can induce the liver transplantation immune tolerance,in order to identify the possibility of adoptive transfer and the specificity of this immune tolerance,we completed this experiment.Objective:To identify the possibility of adoptive transfer and the specificity of liver transplantation immune tolerance induced by donor-derived hepatocytes; Methods:The splenocytes of recipient animals in the first experiment whose surviving time were longer than 20 days were separated routinely,and the splenocytes of animals without liver transplantation were also separated. Sixteen Wistar rats and 8 Lewis rats were used as donors and 24 SD rats were used as recipients,sixteen Wistar rats were divided randomly into two groups and 8 Lewis rats were attributed to the third group.Group A:1ml tolerant animals' splenocyte(2×10~7/ml)were injected into recipients through the vena dorsalis penis,liver transplantation of Wistar rats to SD rats was performed on day 14 after injection.Group B:1ml nulli-transplantation animals' splenocytes(2×10~7/ml) was injected into recipients through the vena dorsalis penis,liver transplantation of Wistar rats to SD rats was performed on day 14 after injection.Group C:1ml tolerant animals' splenocytes(2×10~7/ml)was injected into recipients through vena dorsalis penis,liver transplantation of Lewis rats to SD rats was performed on day 14 after injection.Survival analysis was performed in all three groups.Necrotomy was performed in every died animal in order to obtain the liver for pathological analysis,so as to identify the extent of rejection.Results:The liver time of group A significantly increased compared with that of the group B and the group C,in addition,the grafts damage of recipients whose surviving time were longer than 14 days in group A was more lighter than that of group B and group C.Conclusions:(1)Liver transplantation immune tolerance induced by donor-derived hepatocytes cans adoptive transfer and with specificity;(2)It is possible that the T cells in spleen paly an important action to adoptive transfer of liver transplantation immune tolerance.Part 3The study of the mechanism of liver transplantation immune tolerance induced by donor-derived hepatocytesBackground:The findings of part 1 and part 2 proved that the the immune tolerance of liver transplantation can be induced by donor hepatocytes,and it can adoptive transfer and with specificity.But at the same time,we found a contradictory phenomenon:since the donor hepatocytes evenly distributed along the major branches of the portal veins in the recipient liver,and the recipient liver was removed after liver transplantation,then the donor hepatocytes were also removed accordingly,why the the immune tolerance still be induced successfully? One possible explanation is the donor hepatocytes influenced the immune system of recipient by some mechanism in past two weeks.Objective:To evaluate the effect of fore-treatment of donor-derived hepatocytes on receptor's immune system and approach the induction mechanism.Methods:Wistar rats were sacrificed in order to obtain the hepatocytes and bone marrow cells.Twenty-four SD rats were divided randomly into three groups(n=8),GroupⅠ:1ml donor hepatocytes(2×10~7/ml)were injected into recipients through the vena dorsalis penis.GroupⅡ:1ml BMC(2×10~7/ml)was injected into recipients through the vena dorsalis penis.GroupⅢ:1ml Sodium Chloride was injected into recipients through vena dorsalis penis. Results:Compared with groupⅡand groupⅢ,the level of IL-10 of groupⅠsignificantly higher and the level of TNF-αsignificantly lower.Conclusions:The blood IL-10 level is significantly increased and blood TNF-αlevel is significantly decreased after fore-treatment by donor-derived hepatocytes,the change of the two factors is a possible mechanism of immue tolerance induced by induction mechanism donor-derived hepatocytes.