The Establishment Of Rat Orthotopic Liver Transplantation Acute Rejection Model And Some Related Research

The incidence of acute rejection(AR) of liver allograft is reported to be between 40%and 60%although efficacious immunosuppressive agents have been used.The AR is an immunological process resulting from the recognition of alloantigen by responder cells,during this process,the alloantigen is presented to effector T cells by the MHC-Ⅱmolecular of antigen presentation cells,then the recipient immune system is activated and cytokines are secreted.CD4~+T helper(Th) cells constitute an important arm of the adaptive immune system.Naive CD4~+ T cells could be induced to commit to particular lineages based on mode of stimulation,antigen concentration,costimulation and cytokine milieu.Once differentiated,each lineage was characterized by its own cytokine profile and transcription factors,T-bet for Th1,GATA-3 for Th2,RORγt for Th17 and FOXP3 for induced T regulatory(iTreg).The conventional research thought that the dynamic balance of Th1/Th2 was associated with the prognosis of AR.Activity of Th1 function has been observed to precede rejection in both liver and kidney transplant patients,while Th2 has been noted to be preferentially associated with allograft tolerance.Recently this paradigm has been updated following the discovery of a third subset of Th cells which were known as Th17 cells.Furthermore,iTreg cells were also considered to be immune suppressors.To the best of our knowledge, the roles of Th17 cells and iTreg cells in the process of AR have not been completely elucidated.High throughput genomics and proteomics techniques have facilitated a better understanding of diseases such as AR by deciphering the unique molecular signature that predicts clinical outcomes and therapeutics.An important goal of clinical proteomics is to develop robust,sensitive,and specific methodologies for the simultaneous analysis of all the proteins expressed by the human genome,and to establish bio-signature profiles that discriminate between disease states.Matrixassisted laser desorption,time of flight/ time of flight,mass spectrometry (MALDI-TOF/TOF MS ) and LTQ have been suggested great potential for the early detection of various disease.The objective of this study is to identify biomarkers indicative of prognosis in the serum of rat after OLT. Part One Establishment of Orthotopic Liver Transplantant -on Acute Rejection Model in RatThe objective of this part of research was to establish orthotopic liver transplantation(OLT) acute rejection model in LEWIS to BN inbred Rat,and the influencing factors of success rate of operation and the stability of this model would be analysed,the characteristics distinguish from closed colony rats such as SD and Wistar was summarized.RESULTS:1.The success rate of OLT was 74%(29/ 39),and the ranking of causes leading to the failure of operation were bleeding of suprahepatic vena cava,structural mistake of portal vein,anesthetic accident and the others.2.The problem of body weight could be solved by modifying the technique of OLT, and the using of antibiotics has significantly difference for infection.3.Compared with OLT between closed colony rats,this model had its characteristics.The appearance time,the degree and the result of this acute rejection model did not coincide,however rejection phenomenon appeared fairly well.Part Two The dynamic balance of T-bet~+/GATA-3~+and RORyt/FOXP3~+cells after rat OLTThe objective of this part of research was to To evaluate the dynamic balance of Th1,Th2,Th17 and iTreg cells in the process of AR,OLT was performed from LEW rat to BN rat,and AR was graded using the Banff schema.RESULTS:1.The combination of LEW→BN appeared different severity of AR,while the combination of BN→BN had no evidence for AR.2.The T-bet~+ and RORγt~+ cells were significantly increased in mild,moderate and severe AR group than in control group(P＜0.009;respectively).The GATA-3~+ cells were no difference between mild AR group and control group (P=0.754),while it was significantly higher in moderate and severe AR group (P=0.028 and P=0.009;respectively).The FOXP3~+ cells had no statistically difference(P=0.754),while it was significantly decreased in moderate and severe AR group(P〈0.009;respectively).The ratio of T-bet~+ cells/GATA-3~+ cells was more associated with AR than that RORγt~+ cells / FOXP3~+ cells in the early stage.3.The expression of T-bet~+,GATA-3~+,FOXP3~+ and RORγt~+ in recipient spleen were as following figures.Part Three The proteomic study of rat serum after Orthoto -pic Liver TransplantationThe objective of this part of research was to screen rat serum biomarkers of AR associating with diagnosis and prognosis.Then regulation of those biomarks would be made and the biological effect would be observed.RESULTS:1.A total of 35 proteins were significantly different between control group and AR group(P〈0.01).2.Based on MALDI-TOF/TOF MS and LTQ,30 proteins were identified including hemopexin,Preprohaptoglobin,C3 Complement,C4a Complement C4 precursor, Alpha 1-Macroglobulin Receptor Binding Domain,Mbll Mannose-binding protein,etc.3.The regulation of hemopexin was made and the biological effect would be observed.CONCLUSIONS1.Compared with OLT between closed colony rats,this model had its characteristics.The appearance time,the degree and the result of this acute rejection model did not coincide,however rejection phenomenon appeared fairly well.2.Our study suggested the dynamic change of T-bet~+,GATA-3~+,RORγt~+ and FOXP3~+ cells in the liver allograft and recipient spleen after OLT.Both the T-bet~+ and RORγt~+ cells could have the ability to promote AR,while The GATA-3~+ and FOXP3~+ cells were associated with inhibition of AR,and the ratio of T-bet~+ cells/GATA-3~+ cells was more associated with AR than that RORγt~+ cells / FOXP3~+ cells in the early stage. 3.Based on MALDI-TOF/TOF MS and LTQ,30 proteins were identified including hemopexin,Preprohaptoglobin,C3 Complement,C4a Complement C4 precursor, Alpha 1-Macroglobulin Receptor Binding Domain,Mbll Mannose-binding protein,etc.NOVELTY1.Establishment of a stable model of AR after OLT.2.Investigation the dynamic change of T-bet~+,GATA-3+.RORγt~+ and FOXP3~+ cells in liver allograft and recipient spleen at the first time.3.Some biomarks associated with AR were Screened.