| Part 1 The establishment of acute rejection model after allogenic rat orthotopic liver transplantationObjective To study and reform the technique of rat orthotopic liver transplantation (ROLT), and observe the allograft rejection of liver transplantation for SD→Wistar rat.Methods ROLTs were performed according to Kamada's two cuff technique with some modifications in donor liver acquirement, perfusion, cuff made, recipient anesthesia, anastomosis of suprahepatic inferior vena cava (SHIVC) and shorten anhepatic time. 20 cases who underwent SD→Wistar ROLT were randomly divided into two groups, rejection group and cyclosporine treatment group(CsA group), with 10 cases each. 10 cases who underwent Wistar→Wistar syngenic ROLT were taken for control group. Posttransplantation rejection were observed in each group. Results The time used for total donor operation was 29. 6 ± 5. 2min,trimming of donor liver 20.1+3. 3min, total recipient operation 45.7 ±5.9min, anhepatic phase 17.2 + 2. 4min, anastomosis of SHIVC 10.5 + 2. 2min. More than 93. 3% of the operations were successful. Jaundice, ascites, progressively deterioration of liver function, and finally liver failure could be observed in rats of the rejection group with survival time 5-15 days after allogenic liver transplantation. All of the rats of the control group and CsA group survived more than 1 month. Serum IFN-yand IL-4 level was significantly elevated in the rejection group on the 7th day after transplantation. Both serum IFN- y and IL-4 were at low level in CsA group.Conclusion Posttransplantion survival rate was significantly increased after the reform of surgical technique. The donor liver of SD rats and recipient Wistar rats combination is a high rejection model, and it can be effectively used to study allograft rejection. IFN- y is one of the most important cytokine of liver allograft rejection. As an good index for indication of rejection at early phase of transplantion, high concentration of serum IFN- Y is correlated with the severity of rejection. Activation of T cells and their differentiation from ThO to Thl and Th2 were suppressed by use of CsA, as well as the allograft rejection, but tolerance of the allograft was not induced.Part 2 Recurrence of hepatocellular carcinomar in livergraft: an animal modelObjective To explore a new way to develop a rat model of tumor recurrence in liver allograft with intrahepatic implantation of tumor fragments and the use of immunosuppressant, mimic the progress of clinical hepatocellular carcinoma (HCC) recurrence in liver graft, study tumor recurrence and tumor growth in liver grafts in recipients with HCC under well-defined conditions.Methods CBRH-7919 cell line, which is syngenic to Wistar rat, were cultured in vivo, then implanted subcutaneously in the Wistar rats and serially passaged prior to implantation in the liver. Small tumor fragments of 1X1X lmm were implanted under the liver capsule in the left lateral or median liver lobe. The tumor size was measured with ultrasound under ether anesthesia after 7 days and the tumor volume was calculated. Group A (n = 5), a control group, a tumor was implanted and measured in Wistar livers. In group B (n = 5), a SD-*Wistar allogenic ROLT was performed and a fresh tumor piece was implanted after reperfusion. In group C (n = 5), a tumor was implanted in the liver 7 days after ROLT. Tumor growth curves were compared.Results Tumor take was five of five in group A, one of five in group B, and five of five in group C. A significant growth retardation of the tumor was seen in group B as compared to controls. No difference in growth rate was observed between group C and controls.Conclusion Tumor implantation in the allograft liver 7 days after ROLT under immunosuppression established a safe and reliable rat model of tumor recurrence in liver graft.Part 3 Experimental study on the prevention forpost-transplantation recurrence of hepatocel lular carcinomawith dendritic cells induced donor spleen lymphocytes invitro and in vivoObjective To study the feasibility and validity of adoptive immunotherapy using dendritic cell(DC) activated tumor specific lymphocytes from donor spleen cells to prevent recipient hepatocellular carcinoma(HCC) recurrence after liver transplantation, and to develop an effective method to eliminate residue cancer cells of the recipient, breakthrough the tolerance state to cancer and decrease the recurrence rate of the liver graft.Methods The precursors of dendritic cells were isolated from bone marrow of recipient rats(Wistar), stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4) and cultured for 5 days. The DCs were identified with microscopic morphology and phenotype. Then rat DCs were pulsed with CBRH-7919 tumor cell antigens prepared with freeze-thawed tumor cell lysates. Spleen cells from donor(SD)/recipient(Wistar) rat were cocultured with these DCs respectively to induce CTL as two experimental groups, and unactivited blank donor/recipient spleen cells weremaintained survival by lower dose of rrIL-2 as two control groups. Supernatant IFN- y release were determined by ELISA, and the cytotoxicity of CTL was assayed by LDH release test. 26 cases of posttransplantation HCC recurrence rat model were randomly selected into five groups, 6 cases each. Four groups were treated with adoptive lymphocyte infusion which come from the correspondent groups in vitro, and another group was saline infusion group for blank control. Tumorgenecity, tumor infiltrating-lymphocyte, tumor growth, rat survival, hepatic impairment, rejection, side effect and serum cytokine level were compared between each group.Results Spleen cells activated by DCs loaded with tumor antigen manifested much higher cytotoxic to tumor cells than unactivated group and saline control group. The cytotoxisity of activated cells from donor spleen was significantly higher than that from the resipient. Cytotoxisity to the tumor cells was not observed in both donor and recipient orgin unactivated lymphocytes group. The aggregation of lymphocytes within and near the tumor tissue with necrosis of tumor cells, retardation of the tumor growth, decrease of the tumorgenecity and prolong of the survival time were observed in activated donor spleen cell infusion group without any evidence of liver toxicity and side effect. Some tumor suppression were also observed in activated recipient origin spleen cell infusion group, such as prolong of the survival time and tumor growth retardation, but serum aminotransaminase enzyme was significantly elevated. Conclusion Donor spleen cells infusion which activated by DCs pulsedwith tumor antigen is an practical, safe and effective way to prevent and treat posttransplantation recurrence of HCC. It can decrease postoperative recurrence rate, suppress tumor growth and prolong survival time without any impairment to the liver graft, and it has better treatment and prevention effect than the spleen cells from recipient.
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