| Introduction:The successful development of clinical liver transplantation opens a new chapter for the surgical treatment of liver diseases.At present,liver transplantation has been considered as the only effective method for the treatment of end-stage liver disease,and it is also the best choice for acute fulminant hepatic failure,hepatocellular carcinoma,hilar cholangiocarcinoma and some metabolic diseases With the development of surgical techniques and immunosuppressive agents,the indications of liver transplantation are expanding,and the number of potential beneficiaries is also increasing,as well as a growing shortage of donors.Although livng liver transplantation,organ donation after cardiac death and other marginal donors can alleviate the pressure caused by the shortage of donors to some extent,the problem cannot be fundamentally solved due to the limited number and increasing potential complications.In the long run,the most practical and effective solution is xenotransplantationPig is targeted as the most suitable donor animal due to the relatively similar size of organs to their human counterpart,physiologic similarity,favorable breeding characteristics and feasibility for genetic manipulation.However,pig has clearly more disparities with humans than non-primate mammals,and therefore require a greater degree of genetic modification.Identification of potential genetic epitope targets and their possible roles is crucial to the screening of genetic modification epitopes in transgenic pigsWith the application of various transgenic pigs,pig organ xenotransplantation has made great progress.The survival records of heterotopic pig heart xenotransplantation and kidney xenotransplantation have reached 945 days and 435 days respectively.However,the survival time of pig liver xenotransplantation was still less than a month.Although the cause is not thoroughly identified,it is closely related to coagulation disorders,especially fatal bleeding caused by severe platelet loss.Moreover,anemia was also found to be associated with LXTCurrently,the main reasons for thrombocytopenia after xenotransplantation are considered as follows:(1)the cell surface receptor-ligand interactions between pig endothelial cells or kupffer cells and primate platelets are not coordinated;(2)the presence of natural antibodies to pig liver endothelial antigen leads to the activation of endothelial cells.The effects of asialoglycoprotein receptor-1(ASGR1)and CDllb/CD18(mac-1)are relatively clear in this process.There were also reports showing phagocytosis of human RBCs by pig hepatic phagocytic cells CD47-SIRP?(signal regulatory protein-?)signaling pathway can control phagocytosis by identifying self and non-self.It mediated rejection in phagocytosis experiment in vitro and cell transplantation in vivo experiments,therefore is considered may participate in the thrombocytopenia and anemia after liver xenotransplantation,yet lacking a strong evidence of organ levelAims:Using the mouse liver transplantation model and rat-mouse liver xenotransplantation model to explore the effects of CD47-SIRP? incompatibility on the phagocytosis of blood cells after liver transplantation,thus providing a reliable basis for epitope screening of genetic modification in transgenic pigsMethods:(1)To establish a mature,stable and reliable mouse liver transplantation model by improving the technical details and optimizing the non-technical details.(2)Using a mouse liver transplantation model to investigate the effects of CD47-SIRP?incompatibility on peripheral blood red blood cell and platelet counts after allogeneic liver transplantation.The changes of CD41 fluorescence signal and its colocalization with f4/80 were observed by immunofluorescence staining.(3)To establish rat-mouse liver xenotransplantation,and the existence and types of rejection were determined by histological examination,ALT detection and flow cytometry.(4)Using the rat-mouse liver xenotransplantation model to investigate the effects of CD47-SIRP? incompatibility on peripheral blood erythrocyte and platelet counts in the rejection environment after liver xenotransplantation.Flow cytometry was used to detect platelet activationResults:(1)By optimizing the selection of mouse strain and biliary stent,the long-term prognosis of orthotopic liver transplantation in mice can reach 100%,80%and 80%survival rates at 2 weeks,1 month and 3 months.(2)In the allogeneic liver transplantation model,comparing B6 wild-type(WT)mice after WT liver transplantation,the red blood cell count of CD47KO mice did not decrease significantly,and the platelet count on the postoperative day 1(POD1)and POD5 showed a slight decrease.According to the confocal microscope observation of immunofluorescence staining,the fluorescence signal of CD41 did not increase significantly,and there was no colocalization with F4/80.(3)The rat-mouse liver xenotransplatation model was constructed by using F344 rats with the similar body weight as the recipient mice.Flow cytometry detection confirmed that the main infiltrating cells were CD8+T cells,and the immunophenotypes of T cells were largely transformed from naive and central memory T cells to effector memory T cells.(4)Using the rat-mouse liver xenotransplantation model,there was no significant difference in peripheral blood erythrocytes and platelets change in CD47KO mice compared with WT mice after F344 liver transplantation.The red blood cell and platelet counts of both groups decreased continuously with the passage of time,and there was no significant change in the proportion of activated platelets.Conclusions:(1)The selection of mouse strain and biliary stent restricted the long-term prognosis of orthotopic liver transplantation in mice.(2)CD47-SIRP?incompatibility could not mediate the phagocytosis of blood cells by the liver after allogeneic liver transplantation in mice.(3)The acute severe rejection after rat-mouse liver xenotransplantation was mainly mediated by CD8+T cells.(4)CD47-SIRP? incompatibility could not mediate the phagocytosis of blood cells by liver in the rejection environment after rat-mouse liver xenotransplantation. |
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