The Effect Of Recipient Derived Bone Marrow Mesenchymal Stem Cells Engrafted Into Transplanted Livers After Rat Orthotopic Liver Transplantation

Liver transplantation still remains the only solution for end-stage liver diseases, but acute rejection episodes after liver transplantation was still the main disturbance for long survival.Acute rejection in liver transplantation has been reported to vary between 20% and 70%.It is a frequent cause of hepatic dysfunction in the early phase post surgery. Donor alloantigens induce vigorous immune responses,involving various immune pathways resulting in rapid damage to the graft.Acute rejection is feature of cellularity rejection.Recipient T lymphocyte was acted by donator liver dendritic cells.The recipient activated T lymphocyte aggregated and secreted cytokine. Immunology characteristic was Th1 cells increase secretion of IL-2,IFN-γwhile Th2 cells increase secretion of IL-4,IL-10.One of the Th1 cytokines,IL-2,is thought to be one of the main cytokines responsible for acute allograft rejection.Those cytokine could damag the biliary epithelial cells and vein endothelial cells.Pathobiology characteristic was the admixture lymphocytes infiltration,small biliary ducts and vein vascular endothelial cells had cellular necrosis and exfoliation.The acute rejection came to peak in 7d-14d.CD4+Th cell can differentiate into two groups:Th1 and Th2 cell,each has different function.The balanced state of Th1/Th2 is precondition of the homeostatic immunity.The Th1 cells,which produce IFN-γand IL-2,are dominant in rejection reactions,whereas Th2 cells,which produce IL-4 and IL-10,are involved in the maintenance of immune hyporesponsiveness.Dccrease interferon and IL-4 secretion of TH1 cells and increase secretion of IL-4 and IL-10 from TH2 cells can induce immunological tolerance.The concentration of the Th1 and Th2 cytokines reflect Immuneological station.A Th1 to Th2 shift has the result of immunological tolerance, but a Th2 to Th1 shift has the result of acute rejection.The cytokine environment of liver-resident T lymphocytes and the proportionality between Th1 and Th2 become an index of liver graft immune station.Immunosuppressive drug is the popular method to control acute rejection of liver transplantation,but long-term immunosuppressive drug treatment carries direct toxicity and metabolic side effects.The active induction of allograft tolerance allowing drug-free allograft acceptance with preserved immunocompetence has long been a dream for people.Cellular transplantation has been investigated as a potential therapy for induce immunological tolerance.Mesenchymal stem cells have multipotential for differentiation and immunoregulatory characteristics.Mesenchymal stem cells can mature into elements of many different lineages.The MSCs cultured in vitro exhibited multipotential for differentiation such as hepatocyte-like cells,biliary epithelial-like cells,endothelial-like cells.MSCs also have profound immunomodulatory effects both in vitro and in vivo.The MSCs possess unique immunologic properties.MSCs express negligible levels of human leukocyte antigen(HLA)major histocompatibility complex(MHC) classⅠ.They can be induced to express MHC classⅡantigen,and Fas ligand by interferon-γ(IFN-γ)treatment.Moreover,MSCs do not express co-stimulatory molecules,such as B7-1,B7-2,CD40 and CD40 ligand;therefore,they may not activate alloreactive T cells.MSCs can engraft and survive in a xenogenic immunocompetent environment and maybe induce immunological tolerance.Considering MSCs multipotential for differentiation and immunoregulatory characteristics and other characteristics such as easy taking,simple cultivation.MSCs of recipient are an ideal cell source to induce immunological tolerance.We try to investigate whether MSCs improved liver function and prolonged the survival of transplanted livers and MSCs of recipient origin infused into the liver allograft can differentiate into hepatocyte-like cell,biliary epithelial-like cells,and endothelial-like cells?This study established acute rejection mode of DA to Lewis rat liver transplantation.The recipients were engrafted with donor-derived MSCs by injection through portal vein into livers after the operation.The collected MSCs were labeled by CFSE and BrdU.Detect the labeled MSCs distribution in vivo cell differentiation and the effect on the cytokine secretion of T cells.This study have three chapter.The first chapter:Establishment of rat homogeneity variant mode of orthotopic liver transplantation and pathological detectionAIM:To establish an acute rejection liver transplantation model of DA-to-Lewis rat and detect the characteristics of acute rejection in liver tissues.METHODS:①Eighty liver transplantation models were established by modified Kamada two-cuff technique,with SD rats as the donors and the recipients.No Immunosuppressive drug was gived after operation.The recipients were left to observe the hepatic tissue pathological change on the time of ld,7d,14d of post-transplant and survival time.②Nine acute allograft rejection liver transplantation models were established by modified Kamada two-cuff technique,with the inbred DA rats as the donors and inbred Lewis rat as the recipients.No immunosuppressive drug was gaven after operation.The recipients were left to observe the hepatic tissue pathological change on the time of 1d,7d,14d of post-transplant and survival time.RESULTS:①The successful rate of liver transplantation models(SD liver into SD)was 96%,the long term survival(2mon)rate in recipients receiving orthotopic liver transplantation was 90%.②The liver transplantation models(SD liver into SD)couldn't find immunological rejection in liver tissue.③The liver transplantation models(DA liver into Lewis)can find acute allograft rejection in liver tissue.④The liver transplantation models(DA liver into Lewis)survival time is about 12d to 16d.CONCLUSION:①The liver transplantation models established by modified Kamada two-cuff technique were stably and repeatedly.②The liver transplantation models(SD liver into SD)is not the acute rejection liver transplantation models.The recipients have long-term surviving after operation.③The liver transplantation models(DA liver into Lewis)is the acute rejection liver transplantation models.④The Lewis recipients have short-term surviving after operation without given immunosuppressive drug. The second chapter:Harvesting the recipients derived mesenchymal stem cells and tracking the labeled cells in vivoAIM:To explore a practical method of separation,culture and amplification in vitro of Mesenchymal stem cells(MSCs)from bone marrow of the Lewis rats,and to offer reference to its actual application.To explore a practical method of labeling Mesenchymal stem cells and the distribute variations after infusing into transplanted liver for short time and long time.METHODS:①Ten rat of four week old were sacrificed.Dissected femurs and tibial bones,the bone marrow was eluted with a 4G needle and L-DMEM/F12.The bone marrow mesenchymal stem cells(MSCs)were isolated by combining gradient density centrifugation,split red blood cell and adhesion separation.The 3^thpassage mesenchymal stem cells' biological characterizations such as cell phenotype, generation cycle were detected by Flow cytometric analysis.②The collected mesenchymal stem cells were labeled by CFSE and infused into the transplanted livers through portal vein after the operation.Calculated the labeling index,the MSCs in liver,spleen,lung and kidney were detected by fluorescence microscope on the 1^thweek,2^thweek,1^thmonth.③MSCs were pre-labeled with bromodeoxyuridine(BrdU)before transplantation and infused into the livers through portal vein after the operation.Track the transplanted cells in liver,spleen,lung and kidney tissue by immunohistochemistry stain on the 2^thweek,1^thweek,and 2^thmonth.RESULTS:①The MSCs were spindle shaped,attached to the culture dish tightly after 3 days' culture of primary passage,and proliferated in the culture medium.After 10 to 14 days of primary cultivation,MSCs were nearly 100%confluent.The cells viability detected by Trypanblau was 98%.MSCs were purified at the 3^thpassage.MSCs isolated by split red blood cell were attached to the culture dish more quickly than the MSCs isolated by combining gradient density centrifugation,but proliferated more slowly than the latter.②The 3^thpassage MSCs comprised a unique phenotypic population by flow cytometric analysis.The phenotypes were shown to be positive for CD29(95.3%) and CD44(94.7%)and be negative for hematopoietic markers CD34(98.2%)and CD45(99.2%).③The ratio of cells in G0/G1 stage and S+G2+M stage of the 3^rdpassage MSCs was 82.9%and 17.1%respectively by analysis of generation cycle,which demonstrated that only a part of MSCs remained proliferation ability on a certain stage.④CFSE had high efficiency for labeling MSCs.The labeling index was over 95%.⑤Most CFSE labeled MSCs existed in transplanted liver after operation.Few cells were also detected in other organs,such as lung,kidney and spleen.But the labeled MSCs existed in liver did not reduced much after 14d while that of reducing in spleen, lung,and kidney.⑥The labeled MSCs by CFSE also were infused into the normal Lewis rat.The cells were distributed in liver,spleen,lung,and kidney at 1d,while disappeared at 5d.⑦BrdU had high efficiency for labeling MSCs.The labeling index was over 95%in vitro.⑧Most Brdu labeled MSCs existed in transplanted liver tissues after operation on 14d,1mon,2mon.Few cells were also detected in other organs tissues,such as lung, kidney and spleen.But the labeled MSCs existed in liver did not reduced much after 2 month while that of disappeared in spleen,lung,and kidney tissues.⑨The labeled MSCs by Brdu also were infused into the normal Lewis rat.The cells were distributed in liver,spleen,lung,and kidney at 1d,while disappeared at 5d.CONCLUSION:①Collected MSCs by split red blood cell is simple than that of combining gradient density centrifugation.②MSCs were purified by the method of split red blood cell,combining gradient density centrifugation and adhesion separation.③CFSE stain provides a feasible means for labeling MSCs with high efficiency, most recipient derived MSCs specially existed in transplanted liver and not reduced in a short time.④BrdU provides a simple means for labeling MSCs which could stain for a long time. The recipient derived MSCs specially distributed in transplanted liver for a long time. The third chapter:The effect of recipient derived mesenehymal stem cells engrafted into transplanted livers after rat orthotopic liver transplantationinAIM:To investigate the immigration and affection of recipient derived bone marrow mesenchymal stem cells after orthotopic liver transplantation.The object were included the differentiation of recipient derived bone marrow mesenchymal stem cell in transplanted livers,the effect to survival time and the Th1/Th2 cytokines balance.METHODS:①Thirty DA and thirty Lewis were randomized into 3 groups.A group:only orthotopic liver transplantation,B group:orthotopic liver transplantation + MSCs,C group:orthotopic liver transplantation +CsA,each group have 10 pairs and no immunosuppressive treatment.Hepatic functions and immunological rejection in liver tissue were detected on time of 7d,14d and 1mon.②Each group took liver tissues at the time of 14d,1mon,2mon.The labeled MSCs was detected the co-expression stained with BrdU antibody,ALB antibody,CK19 antibody and vWF antibody by immunohistochemistry double staining.③Each group took liver tissues at the time of 1d,7d,14d,1mon.The expression of IL-2,IL-4,IL-10,IFNγin the liver tissues was detected using the Luminex xMAP system.RESULTS①A group rats were getting bad in 3d after operate and dead in 10-16d.B,C group were deteriorating within the 14d and turned better after 1mon.②Immunological rejection in liver tissues of each group show that the group A showed more serious acute rejection on the time after the operation,but there was little evidence of acute rejection in group B and group C was better than group A. Liver function of B group which had infused MSCs was better than that of A group which had none infused MSCs and that of C group.The difference between B and C group was statistically significant(P＜0.05),There was remarkable difference in survival time between groups A and B and C(13d vs 73d vs 63d,P＜0.05).③The liver tissues of group B had found the co-expression of Brdu and ALB antibody in 1mon and 2mon after operation.The co-expression of Brdu and vWF were also found but not for Brdu and CK19.④The concentrations of cytokines IL-2,IL-4,IL-10,IFNγin each group is higher than the normal group(P＜0.05)in 1d and 7d.But the cytokines concentrations in group B and C were lower than group A relatively.The concentrations of cytokines IL-2,IL-4 in group B were lower than that of group C in 14d,1mon.But the concentrations of cytokines IL-10,IFN-γin group B were higher than that of group C in 14d,1mon.CONCLUSION:①Infused recipient derived MSCs can alleviate the acute immunological rejection after orthotopic liver transplantation.The MSCs-treatment significantly prolonged allografl survival.The MSCs-treatment may be better than the CsA-treatment for the acute immunological rejection after orthotopic liver transplantation.②The recipient derived MSCs which were infused into the transplanted liver had the ability of multipotential for differentiation.The implanted cells could trans-differentiate into various cell types within the transplanted liver such as hepatocyte-like cells and vascular endothelial-like cells.But the bile duct epithelial-like cells labeled with BrdU can not find in the transplanted liver.③MSCs inhibited the cytokines secretion from Th1 and increased the cytokines secretion from Th2 cells,thereby promoting a Th1 to Th2 shift.MSCs maybe induce immunological tolerance after infusing into the transplanted liver.