Study On The Expression Of Ref-1 In Rat Liver And The Relationship Between Its Expression And The Hepatocyte Apoptosis After Liver Transplantation

It is nearly fifty years Since the first successful orthotopic livertransplantation in an adult patient was reported by Starzl. With therapid development of medicine, especially the development of liversurgery in the last 20 years, liver transplant technology steadilyincreased. so that this modality has become established as a standardsurgical method for the treatment of late-stage liver diseases. Atpresent, because of the wide use of efficient immunosuppressants, theincidence of acute rejection reaction has been markedly lowered.Primary graft nonfunction(PNF) became the major cause responsiblefor the early stage liver failure after transplant. There are manyreasons for PNF, however, no one can give a satisfied explanation.The study on the mechanisms for PNF has become an emergent issuefor modern liver transplant surgery.Recent studies showed that the apoptosis is the major cause andappearance for hepatic ischemic reperfusion injury(HIRI) and PNF.HIRI is unavoidable for the graft. This is mainly due to theproduction of ROS which may induce the oxidation and hydrolysis ofDNA bases, breakage of DNA chain. The oxidative damage maycause mutation of DNA bases, interfere with the DNA replication andcell proliferation and at last leads to cell death. Necrosis has beenregarded as the dominant modality for this cell death, however, laststudies have shown that apoptosis was another important deathmodality in this procedure.Ref-1/APE is a wide spreaded multifunction nuclear proteinwhich can repair the injured DNA and indirectly play anti-apoptosisrole by activating diverse anti-apoptosis transcription factors. It is thesole bifunctional enzyme which can repair the DNA AP site in humancells. Many researchers have focused their work on this protein,however, no paper has been published about the relationship betweenRef-1 and apoptosis of hepotocyte after liver transplant.To reduce the impact of immune rejection, we choose thehomologous healthy Wistar rat as the study object for ROLT model.We accurately controlled the cold ischemic time of graft about 40min,ensure the consistence of the ischemic factor of different groups;controlled the anhepatic phase time of acceptor within 20min,guaranteed the achievement ratio of animal model construction wasmore than 98%. The model established substantial foundation.After construction the model of ROLT, we divided the Wistarrats into control group, sham operation and transplant 3h, 6h, 9h, 24hgroup(n=6). The serum biochemics and HE staining were used toobserved function and the morphology change of graft;the TUNELenzyme labeling was applied to observe the change regularity of thehepatocyte apoptosis;immunohistochemistry methods were used toexplore the expression alteration of Ref-1 in graft. All of these wereto investigate the relationship between the hepatocyte apoptosis inearly stage of ischemic/reperfusion and the Ref-1 change regularity.Further to probe that whether the Ref-1 could relieve HIRI.The results showed:1, the ROLT model constructed with modified doublecatheterization cannula was the ideal animal model for liver transplantwhich had excellent stability and reproducibility. High-grade graft,skilled implantation technique and consummate perioperativemanagement were the key questions for model construction.2, the hepatocyte apoptosis began to increase at 3h aftertransplant, got the peak level at 12h and slightly decreased at 24.3, apoptosis was the main modality of cell death at the early stage.The degree of apoptosis reflected the degree of injure.4, the expression of Ref-1 decreased after transplant and got thelowest at 24h, showing the time dependent effect.5, the Ref-1 positive staining cells gradually decreased while theTUNEL positive staining cells gradually increased at the same studypart. This result indicated that the down-regulate of Ref-1 andsubsequent breakdown of DNA repair may be an important reason forapoptosis accumulation.We successfully set up the ROLT model with excellent stabilityand reproducibility, revealed the change regularity of Ref-1 at theearly stage of liver transplant, observed the apoptosis of hepotocyte,verified that the apoptosis was the main modality of cell death at theearly stage of transplant, indicated that the down-regulation of Ref-1may be responsible for apoptosis. We will apply the adenovirus asvector, administrate exogenous Ref-1 through the portal vein andobserve its effects on ischemic/reperfusion injury after transplant.The further study will contribute to understanding the relationshipbetween Ref-1 and the apoptosis. The results may be useful fortreating ischemic/reperfusion injury, protecting graft and prolongingthe survival time.