The Study Of PD-L1Ig And Anti-CD40L Monoclonal Antibody Inducing Immunotolerance In Rat Liver Transplantation

Liver transplantation has become an effective treatment for the patiens with final liver diseases.But, the rejection after transplantation is the main factors influencing the long-term surviving of patients undergoing liver transplantation.Therefore, the best way to overcome the transplant rejection is to explore a new method of inducing immune tolerance. In tolerance induction method,more and more scholars pay close attention to common stimulate signal pathways which have become a hot spot of the immunology. In common stimulate signal pathways, PD-1/PD-L signal pathways are regarded as close relative with the transplant rejection. Since the ideal results of inducing immunotolerance cannot be obtained by blocking single pathways among common stimulate signal pathways, many scholars also belive that the desired results of inducing immunotolerance will be better obtained as blocking many common stimulate signal pathways. In recent years, after CD28,CD40-CD40L pathways have received much attention once more. Many research show that the PD-1/PD-L pathways and CD40-CD40L pathways have an interactive function. This experiment aims to study that PD-L1Ig and anti-CD40L monoclonal antibody work together to influence on inducing immunotolerance of rat liver transplantation base on establishing stable rat liver transplantation model.PartⅠThe establishment of rat orthotopic liver transplantation modelObjective To explore that the key point in the experiment of orthotopic liver transplantation(LT) in rats lies in establishing a stable and reliable animal model.Methods LT model using Kamada's two-cuff technique was established with healthy clean adult Wistar and Sprague Dawley (SD) rats. Results Twenty cases have been carried out on the rat LT.16 rats survived more than 7 days after operation.Thus, the success rate was 80%,which accords with the demand of this experiment. The rest of 4 cases failure to survive more than 7 days:1 case died of anesthesia accidents,1 case died of intestinal obstruction,2 cases died of the suprahepatic inferior vena cava anastomotic bleeding. Histopathologic examination of their liver were performed on 7 days after sugery.A large amount of inflammatory cell infiltration was determined in the portal tracts, inflammatory of vein endothelial in the portal regions, bile duct injury, degeneration and necrosis of hepatocytes severe rejection.Conclusion These experiments all prolong the survival time of rats, which need the improve of skilled surgical operation, the meticulous surgical performance and some effective steps to remedys.Thus, the stable and reliable animal model was established, the model could be used to study immunotolerance.PartⅡThe study of PD-L1Ig and anti-CD40L monoclonal antibody inducing immunotolerance in rat liver transplantationObjective To explore the role of PD-L1Ig and anti-CD40L monoclonal antibody working together in inducing immunotolerance after liver transplantation. Methods After liver transplantation., the rats were divided into four groups:normal control group(A group), anti-CD40L antibody group(B group),PD-L1Ig group(C group), anti-CD40L antibody and PD-L1Ig group(D group). The control group:only transplantation. B group were injected with PD-L1Ig0.1mg/kg by intraperitoneal injection,C group were injected with anti-CD40L antibody0.2mg/kg,D group were injected with PD-L1Ig 0.1mg/kg and anti-CD40L antibody 0.2mg/kg.The medication were given on the day of 1,2,4,6 after LT. After operation, the therapeutic effects including general condition of rats such as survival condition and appetite were evaluated and compared among four groups. The serum ALT,AST and TBIL level were detected after operation in a week later and Hepatocyte apoptosis were analyzed using flow cytometry. Then HE stain for liver were used to evaluate the severe rejection of liver histology.Results The lifetime of PD-L1Ig and anti-CD40L monoclonal antibody working together is twenty four days,which is the longest in four groups, there is significant difference(p＜0.05), B group and C is longer than A group(p＜0.05),B group and C group showed no significant difference (P＞0.05).;The therapeutic effects including general condition of rats such as survival condition and appetite in A group were significantly worse than B,C,D group. The serum ALT,AST and TBIL in A group were higher than the three group above (P＜0.05). The serum ALT,AST and TBIL in D group were lower than B group and C group (p＜0.05). B group and C group showed no significant difference (P＞0.05).7 days later after operation,rats liver periportal accumulated a large number of mononuclear cells in control group by Liver pathology detected. A large amount of inflammatory cell infiltration was determined in the portal tracts, inflammatory of vein endothelial in the portal regions, bile duct injury in B group and C group. The periportal inflammation of the liver in D group was relatively light, only a small amount of the accumulation of inflammatory cells. The liver rejection of the control group was severer than that of D group on the basis of referencing Baff marking. The liver rejection of B group and C group were severer than that of D group on the basis of referencing Baff marking. Hepatocyte apoptosis were analyzed using flow cytometry showed that the transplanted liver apoptosis rate of D group lower than the three group above.Conclusions 1. PD-L1Ig and anti-CD40L monoclonal antibody working together to reduce graft rejection is better than that only using PD-L1Ig or anti-CD40L mAb.2. After liver transplantation, PD-L1Ig and anti-CD40L monoclonal antibody working together can significantly prolong the survival time of rats,and the effect of improving liver function for PD-L1Ig and anti-CD40L monoclonal antibody working together is better than that only using PD-L1Ig or anti-CD40L mAb.3. PD-L1Ig and anti-CD40L monoclonal antibody working together reduce liver cell apoptosis is better than that only using PD-Lllg or anti-CD40L mAb.