Experimental Study For Apoptosis Of Activated T Cells To Induce Spontaneous Tolerance Of Rat Liver Allografts

Part one ESTABLISHMENT OF SPONTANEOUS IMMUNE TOLERANCE MODEL IN INBRED RAT LIVER ALLOGRAFTSObjective:To choose inbred BN and LEW combinations to establish spontaneous immune tolerance model in rat liver allografts so as to provide animal models for performing the experimental study on immune tolerance of liver transplantation.Methods:Surgical skills were trained by using closed colony Wistar rats,followed by orthotopic liver transplantation between BN and LEW rat combinations with Kamada's two cuff technique.Experimental animals were divided into three groups,namely A group,syngeneic transplantation between LEW and LEW rats as recipients,B group, allogeneic transplantation between LEW and BN rats as recipients and C group,allogeneic transplantation between BN and LEW rats as recipients.The grades of acute rejection were judged by their clinical manifestations and Banff schema.Four cases of cervical heterotopic heart transplantation were performed between BN rats as donors and two recipients of C group and B group sixty days after liver transplantation,respectively,in order to identify whether the transplant hearts were rejected.Results:171 models of rat liver transplantation were established,the total success rate being 60.8%(104/171).The success rate was only 17.1%(12/70)during the protophase of model manufacture.Afterward with the operation skills improved,the success rate gradually comes up to more than 90%,The causes of death were anesthetic accident,hemorrhage shock,pulmonary edema, thrombosis and stenosis of suprahepatic and infrahepatic vena cava and primary graft nonfunction.In recipients of A group,liver function injury and interstitial edema upon histology examination occurred within one week after liver transplantation,but rejection was not founded.In recipients of B group,I grade rejection occurred at 3rd day and rejection gradually reached the peak 9 days after transplantation,and all died ofⅢgrade rejection within 14 days after operation.ItoⅡgradere jection also appeared in recipients of C group from 5rd,but the rejection did not progressively get worse,tissue structure of allografts recovering to normal 28 days after liver transplantation.The cervical heterotopic heart transplantation results showed that two recipients of C group 60 days after liver transplantation did not reject the hearts from BN rats as donors,but two recipients of A group 60 days after liver transplantation repelled the hearts from BN rats as donors at 7th and 9th day after heart transplantation,respectively.Conclusion:(1)Better operation skills and the attention to details concerned with operation are the key points to establish liver transplantation models successfully.(2)The liver transplantation combinations between BN to LEW rats can produce stable spontaneous immune tolerance,which is estimated to occur sixty days after operation,and is thought to be a better animal model for studying immune tolerance of liver transplantation.(3)Compared with outbred rats,the liver transplatation model between inbred rat combinations is more difficult to establish because of their tissue characteristics and poor operation toleration.Part two EXPERIMENTAL STUDY FOR APOPTOSIS OF ACTIVATED T CELLS TO INDUCE SPONTANEOUS TOLERANCE OF RAT LIVER ALLOGRAFTSObjective:To choose inbred BN and LEW combinations to establish spontaneous immune tolerance model in rat liver allografts at first,and then,utilizing flow cytometry,immunohistochemistry and molecular biology technique to explore the role for apoptosis of activated T cells and immunomodulation which was induced by the apoptosis in spontaneous immune tolerance of rat liver transplantation so as to provide theoretical basis for the induction of immune tolerance of clinical liver transplantation and reasonable utilization of immunosuppressive agent.Methods:Healthy,clean and male inbred BN and LEW rats were randomly divided into four groups:A group(n=15),allografts between BN rats and LEW rats as recipients,Bgroup(n=15),allografts between LEW rats and BN rats as recipients,C group(n=15),allografts between BN rats and LEW rats as recipients to whom Methylprednisolone was administered by a dosage of 12mg/kg on the operation day and 8mg/kg on the first day after operation,respcetively,and D group,isografts between LEW rats and LEW rats as recipients,orthotopic liver transplantation models between BN and LEW rat combinations were established with Kamada's two cuff technique.Recipients were sacrificed at 3rd,5th and 7th day postoperatively(n=3 animals/group/time point for A,B and C group,n=2 animals/time point for D group).Representative liver allografts tissue and serum specimens were collected for corresponding detection.Meanwhile some fresh allografts tissue specimens were snap frozen in liquid nitrogen for fluorescence quantitation PCR detection of Fas,IL-10.TGF-βand Foxp3 gene.Remaining recipients were observed for long-term survival.Results:(1)57 models of rat liver transplantation were established,the total success rate being 91.2%.The survival rate analysis indicated that the life span of A and D group was significantly longer than that of B and C group(P＜0.01),the prognosis of B group being poorest among the whole groups;(2)Apototic T cells appeared in liver allografts of A group 3rd day after liver transplantation,reached the peak 5th day after operation and subsequently descended,whereas less apoptotic lymphocytes emerged in liver allografts of B,C and D groups.Apoptotic lymphocytes rate was significantly higher in liver allografts of A group than in that of B,C and D groups(P＜0.05);(3)The serum level of IL-2 and IFN-γof A group heightened very distinctly 3rd day after operation and Subsequently gradually falled off.The serum level of IL-2 and IFN-γof A group 3rd day after operation was extremely different from that of B,C and D groups(P＜0.01).The serum level of IL-2 and IFN-γof B group slowly stepped up postoperatively and reached the peak 7th day after operation;(4)There was no difference of proportion of CD4+CD25+Treg among all groups within 3 and 5 days after operation,but the ratio that CD4+CD25+Treg occupied CD4+ cells was significanty higher in A group 7th day after operation than that in B,C and D group;(5)Fas gene was highly expressed postoperatively in A group,reached the peak 5th day after operation and subsequently decreased.By statistical analysis,the relative expression of Fas gene in A group 3rd and 5th day after operation was significantly different from that in B,C and D groups(P＜0.05);(6)There was no difference of relative expression of IL-10 and TGF-βgene among all groups within 3 and 5 days after operation,but the relative expression of IL-10 and TGF-βgene was significanty higher in A group 7th day after operation than that in B,C and D group(P＜0.05);(7) There was no difference of expression of Foxp3 mRNA among all groups within 3 and 5 days after operation,but the relative expression of Foxp3 gene in A group 7th day after operation was significanty different from that in B,C and D group(P＜0.05),suggesting that the expression of Foxp3 gene begin to strengthen from 7th day after operation.Conclusion:(1)Apoptosis of activated T cells,which may be implemented by Fas death receptor path,plays important role in spontaneous immune tolerance of rat liver allografts;(2)CD4+ CD25+ Treg may be one of factors that induce spontaneous immune tolerance of rat liver allografts,and the immunomodulation function of CD4+ CD25+ Treg may be concerned with the relative expression of Foxp3, IL-10 and TGF-βgene;(3)The immunomodulation that is induced by apoptosis is intimately associated with the long-term maintainance of spontaneous immune tolerance;(4)Administration of large dose of steroid hormone in the middle of operation or during early stage after liver transplantation may be disadvantageous to the formation of long-term immune tolerance by inhibiting apoptosis of infilitrating lymphocytes in liver allografts.