| Part I Establishment of rat orthotopic liver transplantation model and the choice of different strainsObjective : To build experimental model of orthotopic liver transplantation in rat(ROLT),summarize operative skills and technique, observe the development process and basic pathophysiologic changes of acute rejection(AR)following allotransplantation from Lewis(LEW)to Brown Norway(BN)rat and Sprague-Dawley(SD) to Wistar rat.Methods: The rats were divided into two groups, SD→Wistar group, Lewis→BN group , The orthotopic liver transplantation model was established by Kamada's two cuff techniques with some modification,of which suprahepatic vena cava(SHVC)was anastomosed with continuous suture,portal vein(PV)and infrahepatic vena cava(IHVC)were anastomosed with cuff method,and the bile duct was reconstructed by interior stent. Recipients were sacrificed on 3d,5d,7 d postoperatively and liver tissues and blood samples were collected. Recipient survival rate, operative achievement ratio, complication, histopathological characteristics were observed, Plasm levels of alanine aminotransferase(ALT), aspartate aminotransferase (AST)total bilirubin(TBIL) were measured with automatic biochemical analyser.Results: There was no significant difference of procedure time between the two operation groups(P > 0.05). The 48 hours survival rate of closed colony group was 93.3%,and inbred group was 90.0%, there was no significant difference between the two operation groups(P > 0.05). The causes of death in the operation were deep anesthesia, obstruction of respiratory tract, hemorrhagic shock. The causes of death within 24 hours after operation were bled in the vessel anastomotic stoma, thrombus formation. The one month survival rate of closed colony group was 83.3 %,while the inbred rat died within 11d, there was significant difference between the two operation groups(P < 0.05). The causes of death beyond 24 hours after operation were obstruction of biliary tract, infection. The histopathological change of closed colony group in 3d, 5d, 7d after operation were Williams grade 2, grade 1, grade 1 acute rejection standard respectively. The histopathological change of inbred group in 3d, 5d, 7d after operation was Williams grade 2, grade 2, grade 3acute rejection standard respectively. The AST, ALT and TBIL level of closed colony group decreased after operation, and the histopathologic changes of liver tissues were relieved gradually. The hepatic zymogram and Williams grade of inbred group increased gradually after operation and were higher than closed colony group.Conclusions: The sophisticated microsurgical techniques and the delicate surgical manipulation is the key to successful establishment of rat orthotopic liver transplantation model. we improve Kamada's two cuff technique in three points: segregate liver graft anticlockwisely; perfuse through abdominal aorta; excise diaphragmatic muscle annulatio when trimming suprahepatic vena cava. SD→Wistar combinations is a tolerant model, which is not suitable for the study of acute rejection of liver transplantation in practically and theoretically. Lewis→BN combinations is an ideal rejection model and can be used for basic study on transplantation immunity. The pathological changes and clinic characters were consistent with the standard of acute rejection in liver transplantation.Part II Effect of Heat Preconditioning of Rat Liver Graft on theCytokine ANDγδT of RecipientsObjective: To study the effect of heat preconditioning of rat liver graft on the Cytokine,γδT of recipients.Methods: The rats were randomly divided into two groups, control group and heat preconditioning group. Recipients were sacrificed on 3d, 5d, 7d, 12d after liver transplantation and liver tissues and blood samples were collected. Recipients survival rate, histopathological were observed, Plasm levels of ALT, AST, TBIL were measured with automatic biochemical analyzers. IL-2, IFN-γ,IL-4 and IL-10 in plasm were assayed by ELISA , mRNA expression of cytokine in liver tissues was detected with reverse transcription-polymerase chain reaction(RT-PCR), TheγδT infiltrated in hepatic allograft was detected by Flow CytometryResults: The rejection grade of both groups increased gradually after operation and control group was severe than homeochronous HP group. The hepatic zymogram of both groups increased gradually after operation and control group were higher than homeochronous HP group (P<0.05) . The infiltrating inflammatory cell of control groups were severe than homeochronous HP group (P<0.05) . The IL-2,IFN-γconcentration of both groups increased gradually after operation and control group was higher than homeochronous HP group (P<0.05) , There was no difference in IL-10 concentration between two groups (P>0.05) .IL-4 was not detectable in two groups. The relative expression of IL-2,IFN-γmRNA of both groups increased gradually after operation and control group was higher than homeochronous HP group (P<0.05), There was no difference in the relative expression of IL-4,IL-10 mRNA between two groups (P>0.05) .TheγδT ratios of HP group was higher than homeochronous control group (P<0.05). The MST of control group was 9 days. HP group was 16 days. The survived time of HP group was longer than control group (P<0.05 ) . Conclusion: Heat preconditioning elevated theγδT ratios in allograft mononuclear and delayed the ascensus of Th1 cytokine, but had not manifest effect on Th2 cytokine. Heat preconditioning extenuated the grade of infiltrating inflammatory cell. There was not relation between cytokine andγδT after transplantation. Heat preconditioning may prolonged the survived time of recipients.Part III Study on the HSP60 Prolong The Survival Time of Murine Skin AllograftObjective: To investigate the influence and mechanism of HSP60 on the murine skin allografts.Methods : 8～12 weeks old, inbred, female BALB/C(H-2~d) mice(n=45),CBA/N (H-2k)mice were used as transplantation donors and C57BL/6(H-2~b) mice as recipients. Recipients C57BL/6 mice were devided into 4 groups randomly. Group A: 1cm×1cm Wolfe-Krause skin graft were excised from the back of BALB/C mice and hypoderma were scraped off aseptically, then transplanted to the back of C57BL/6 mice. The methods of skin transplantation of other groups were similar to group A. Group B: C57BL/6 mice were treated with IFA administration into the back 2 weeks before transplantation of BALB/C mice skin. Group C: C57BL/6 mice were administered HSP60 emulsified in IFA into the back 2 weeks before transplantation of BALB/C mice skin. Group D: C57BL/6 mice were treated with HSP60 emulsified in IFA administration into the back and followed by skin transplantation of CBA/N mice 2 weeks later. The delayed type hypersensitivity was determined at 7d after transplantation. One way mixed lymphocyte reaction, the concentration of cytokines in the mixed lymphocyte reaction culture supernatant were determined at 7d, 25d after transplantation. The survival time of skin allograft was observed.Results: The survival time of skin allograft of A,B,C,D groups were 12.4±0.5d,11.6±0.8d,28.3±2.6d,26.4±2.1 d, there was significant difference between A,B groups and C,D groups (P < 0.05) ,while there was no difference within the A,B group and within the C,D group (P>0.05 ) .The cpm of mixed lymphocyte reaction in 7d after transplantation of A,B,C,D groups were 12836±1357,11876±1265,6581±573,6843±612, there was significant difference between A,B groups and C,D groups (P < 0.05) ,while there was no difference within the A,B group and within the C,D group (P >0.05) . The cpm of mixed lymphocyte reaction in 25d after transplantation of A,B,C,D groups were 13286±1498, 12960±1376 , 11936±1265 , 12374±1269, there was no difference between the A,B, C,D group (P> 0.05) .The concentration of IL-10 in the mixed lymphocyte reaction culture supernatant of C,D groups was higher than A,B groups, and IL-2,IFN-γwere lower than A,B groups in 7d after transplantation (P < 0.05),while there was no difference within the A,B group and within the C,D group (P>0.05) . There were no difference in cytokines among 4 groups in 25 days after transplantation (P>0.05) . The delayed type hypersensitivity of A,B, C,D group in 7d after transplantation were 0.84±0.09 mm,0.81±0.07 mm,0.43±0.05 mm,0.46±0.03 mm, there was significant difference between A,B groups and C,D groups (P < 0.05) .while there was no difference within the A,B group and within the C,D group (P > 0.05) .Conclusion: HSP60 may play a role in the induction and maintenance of murine skin allografts tolerance. |
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