| BackgroundOrthotopic liver transplantation(OLT) is a routine and most effective clinical procedure for patients with end-stage liver failure.However,despite standardization of surgical techniques and continuous refinements in anti-rejection therapies,long-term results of transplantation have not significantly improved.Immunosuppressive drugs and regimens has made organ transplantation become an accepted therapeutic modality for end-stage renal,liver,and heart disease and significantly reduced the incidence of acute rejection and resulted in ever-increasing rates of short and long term organ allograft survival.Unfortunately,many problems occurred after new effective immunosuppressive drugs were used.First,immunosuppressant must be continued for life and nonspecifically suppresses the immune system.Secondly,none of the numerous immunosuppressive agents that are currently available can abolish completely the risk of acute and especially chronic rejection.Thirdly,their use bears the risk of malignancies and infections.Finally,all transplant patients are at increased risk for cardiovascular complications related to hypertension and drug-induced elevation to serum levels of cholesterol and triglycerides.Therefore,induction specific immunologic tolerance is the ideal method to solve problem about rejection. Contrast to immunosuppressive treatments,induction specific immunologic tolerance is superiority,for avoiding rejection fundamentally and not affecting normal function of anti-infection and immunologic surveillance.Meanwhile,induction specific immunologic tolerance is the ideal method to solving problem about rejection of xenotransplantation,because of immunosuppression of xenotransplantation need long-term,high-level,nonspecific immunosuppressive agent to maintain.So, inducing immunotolerance,handling graft rejection and preserving graft's funtion is the extremely significant subject in fields of allotransplantation or xenotransplantation.Though there are so many means to induce the tolerance,none of them can achieve the complete permanent specific tolerance.Apoptosis is a kind of physiological or pathological phenomenon generally existing in the biosystem,and plays an important role in maintaining the balance and stabilization of the host's immune state.Apoptotic cells can secrete lipidic haemostatic factors,and present "eat-me" signals on the surface to attracted the phagocyte to remove themselves;at the same time,antigent-presenting cells(monocytes and macrophages,etc) can secrete many inhibitive immune factors such as TGF-β,PGE2,IL-10 etc,after the phagocytosis.All of these creat a microenvironment for antigen recognition and induce the related lymphocytes' tolerance to apoptotic antigen rather than the inflammatory response.On these grounds,Sun et al proposed to make use of the transfusion of apoptotic cells to induce the donor's specific tolerance.Cytokine is a cell-to-cell signaling peptide generated by Activated cells.Most cytokines have multiple sources,multi-role and multi-target effects.Cytokine network is not only an essential component of immune recognition and response,but also one of the main factors in deciding the direction of immune response,a variety of reaction Change caused by Antigenic substances into the body's immune system include the change of the proportion of cytokine.Because the latter directly participate the occurrence process and the final outcome of the former,exploring the change of cytokine have significance in analysis and prediction of the immune response.Our workgroup has demonstrated that a series of heart or liver transplantation rat models that immune tolerance and long-term survival are achieved in recipients by pre-transfusion of donor apoptotic splenocytes.And we find that the donor apoptotic splenocytes are mainly trapped into liver after transfusion with the tracking in vivo. And the cytokine microenvironment of liver is affected by it.But whether it affect the cytokine microenvironment of recipient peripheral environment.So based on the fore-experiment,we resolve that whether affect the microenvironment of peripheral blood of donor apoptotic splenocytes transfusion.ObjectiveTo investigate the effects of donor apoptotic splenocytes transfusion on peripheral blood cytokine microenvironment.MethodsDonor,14 C57BL mice,male,8-10 wks old;Recipient,50 Balb/c mice,male,6-8 wks old.The following examinations are performed:(1) Spleen cells are separated by mechanical trituration,and lymphocytes are separated by yide separating medium.(2) Apoptosis of donor splenocytes is induced by U.V.,and the rate of apoptosis detected by flow cytometer.(3) Using intravenous transfusion,the following examinations are performed in every 5 mice at each time point for each treatment group.Grouping:living cell treatment group(LP) and apoptotic cell treatment group(AP);(4) The time of test:0h,1h.3h,6h,12h;(5) Collecting sample by Cardiac puncture in vitro;(6) Cytokines are assayed by Luminex liquid array:IL-1β,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,GM-CSF,IFN-γ,TNF;(7) The data are statistically calculated and diagrammed by SPSS 13.0 and GraphPad PRISM 3.02.Statisticsexperimental data expressed as mean±SD,General Linear Model-Univariate, One-way ANOVA,Independent Samples t-Test,analysis were used to estimate the item differences among the groups using SPSS13.0 for Windows.And graph was maken by GraphPad PRISM 3.02.Results(1) The proportion of apoptotic induced by U.V.is more than 41.07%which detected by flow cytometry;(2) Changes of cytokines in LP(IL-1β,IL-2,IL-4,IL-5,TNF can not be tested for the low content):IFN-γis higher at 6h(P=0.010,6h),IL-10 is higher at 3h and 6h(P=0.009,3h;P=0.015,6h),IL-12,GM-CSF and IFN-γhave no significant difference at each time point.(3) Changes of cytokines in AP:IL-6,IL-10,IL-12,IFN-γhave no significant difference at each time point.GM-CSF is higher at 3h(P=0.049).(4) Main effects of treatment on cytokines;①IL-6:The differences between LG and AG are statistically significant(P= 0.035,1h).Between them,at 1h after transfusion,LP have higher amount of IL-6 than AP.②IL-10:The differences between LG and AG are statistically significant (P=0.001,3h;P=0.001,6h;P=0.043,12h).Between them,at 3h,6h,12h after transfusion,LP have higher amount of IL-10 than AP.③IL-12,GM-CSF,IFN-γ:The differences between LG and AG are not statistically significant.ConclusionHowever Experiments show that IL-6 is statistically significant at 1h and IL-10 is statistically significant at 3h,6h,12h,LP have higher amount of IL-6 and IL-10 than AP,other cytokines have no significant difference,and after the infusion of Living cells,3h,6h of IL-10 concentration was significantly higher than 0h.And after transfusion of apoptotic cell,the cytokines haven't changed basically.From the result of IL-6 and IL-10,we can speculate:After infusion of living cells,the acute inflammatory response is generated in body,making IL-6 increased at 1h,and after three hours,in order to avoiding the excessive of inflammatory response in the body, the protective measures have taken to increase the secretion of IL-10 which can block synthesis of IL-6,TNFαand other media in monocyte and macrophage.however apoptotic cells had no significant role in the peripheral blood.Because apoptotic cells is a dead cell,having a complete envelope and clean-up with phagocytosis,the inflammatory response is weak in relation to the living cells.According to previous studies,apoptotic cells of the body have a clear role,can leading to immune tolerance, and the group's preliminary studies of donor apoptotic splenocytes tracking in vivo found that the donor apoptotic splenocytes are mainly trapped into liver after transfusion and can affect the cytokine micro-environment in liver,thus we can speculate that the apoptotic cells play a mainly role in the local:liver,However,the specific mechanisms need to be further researched on other cell types and change of response function of liver tissue,such as memory T cells,B cells etc.
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