Immune Hyporesponsiveness Induced By Application Of Recipient-Derived Immature Dendritic Cells In Rats Livers Transplantation

Immune Hyporesponsiveness Induced by application of Recipient-Derived immature Dendritic Cells in rats livers transplantationPartⅠEstablishment of acute rejection model of orthotopic liver transplantation in ratsObjective:To establish an acute rejection model of orthotopic liver transplantation(OLT)in the rats and observe the characteristics of acute rejection.Methods:1 Surgical skills were trained by using closed colony SD rats,modified two cuff(portal vein and infrahepatic vena cava)technique was applied for the model of orthotopic liver transplantation in rats.2 Compared with the survival rate,complication and pathologic damage was observed in the two groups between SD-wistar and Wistar-Wistar rats after liver transplantation,every 5 recipients were sacrificed respectivelp at day 4,7,10 after OLT to detect the changes of serum biochemistrp ALT(alanine aminotransferase,ALT)and TBIL(total bilirubin,TBIL)and the grafts histology,the other 5 recipients were left in each group to observe the animal survival.Results:1 The animal survival time was no more than 72 hours but experiences were accumulated in stage of Surgical skills were trained.2 ALT values in SD-Wistar group was progressive advanced but gradually stepped down in Wistar-Wistar group,There was significantly difference(P＜0.01)in two groups at the same tome.3 Compared with Wistar-Wistar group,TBIL values in SD-Wistar group was progressive advanced at day 7,10(P＜0.01).4 In SD-Wistar group,the histological manifestations of the grafts were obvious rejection at day7,and became more sever at day 10.The Banff scale of grafts in SD-Wistar group wasⅡtoⅢgrade rejection but in Wistar-Wistar group was 0 grade rejection at day 10.Conclusion:1 The model of rat orthotopic liver transplantation is a complicated procedures.It needs careful and patient treatment in operation and perioperation.2 The acute rejection of SD to Wistar orthotopic liver transplantation model is stable and intensive,It can be effectively used to study allograft acute rejection. Methods:Fresh bone marrow cells(BMC)harvested from rat femurs and tibias were treated with hypotonic buffered tris-ammonium chloride to depleted of red blood cells.The cells from bone marrow precursors were cultured in gelatin coated cultrure dishes with recombinant rat granulocyte-macrophage colony-stimulating factor(rrGM-CSF)and interleukin-4(IL-4).The cells were divided into two groups(mature group and immature group)at day 5.The mature DCs group(IL-4 group)were stimulated with complete culture medium containing granulocyte-macrophage colony-stimulating factor(20ng/ml)and Interleukin-4(10ng/ml)last 4 day of the culture,but the immature DCs group(IL-10 group)were culture with IL-10 10ng/ml added to the culture medium.The loosely adherent cells in both groups were collected for experiment at day 9.Cells morphological differences between two groups were observed daily by optical microscope and scanning electron microscope.Cell surface molecules including MHC-Ⅱ,OX62,CD80 and CD86,were assayed by flow cytometery(FCM).The two groups of DCs were cocultured with the allogeneic T lymphocyte.The proliferation of T lymphocytes in mixed leukocyte reaction(MLR)and CD4~+CD25~+Tre lymphocytes were examined through method of MTT and FCM respectively.To collect the culture medium of the two groups at different time during the culture,The Level of IL-12 Secreted by DC were measured by enzyme linked immunosorbent assay(ELISA) kit to evaluate the relationship between the IL-12 secrection and the maturation state of DCs.Results:1.Morphologic features of harvested dendritic cells:Compared with mature DC,IL-10 group DC was smaller and less of obvious sheet like processes and veils.2 Cells expressing OX62 in IL-10 group were almost the same as IL-4 group (88.60±4.22%vs 89.46±5.94%,P＞0.05),which indicates the high purity of DC was harvested in both groups.Expression of MHC-Ⅱmolecule in IL-10 treated DC was lower than in mature DC(19.62±2.30%vs83.46±6.32%,P＜0.01).Expressions of costimulatory molecules,such as CD80 and CD86 in IL-10 group DC were significantly lower than mature DC(19.89±2.48%vs81.98±6.30%,20.59±3.21%vs88.18±5.72%,P＜0.01)2 MLR showed that IL-10 group DC had weaker activity of stimulating proliferation of allogeneic T cells than mature DC at different concentration of stimulating cells(0.304±0.052 vs0.669±0.251,0.52±0.084vs1.144±0.125,0.534±0.079vs1.547±0.136,0.624±0.078 vs 1.944±0.112,P＜0.01).These immature DC were poorer inducers of allogeneic T cell activation.3 The percentage of CD4~+CD25~+ T lymphocytes were increased by stimulation of either group of DCs.The DCs of immature group seem to be more inducer than the DCs of mature(P＜0.01).4 DC secrected IL-12 in a time-dependent manner,the level of IL-12 in mature group is significantly higher than that in immature group(P＜0.01).Conclusion:1 IL-10 can inhibit maturation of bone marrow-derived dendrite cells,and immature dendritic cells can induce allogeneic immune Hyporesponsiveness in vitro.2 Evaluation system has been established to identify the immature state of DC,which involved morphology,phenotype,the production of IL-12 and the effects of DC on T cell including the stimulatary potential,the cytokines secretion and the percentage of CD4~+CD25~+T cell.3 A reliable method to generate large number of DCs from rat bone marrow in vitro has been established.PartⅢThe Mechanism of immune Hyporesponsiveness Induced by application of Recipient-Derived immature Dendritic Cells in rats livers transplantationObjective:To study the mechanism of immune Hyporesponsiveness of allograft rejection induced by transfusion nonpufsed allopeptide syngeneic immature Dendritic cells generated from recipient bone marrow progenitors,explore a possible strategy for liver transplantation allograft protection in clinic.Methods:1 Immature dendritic cells(imDC)generated from recipient rat were prepared for use before transplantation.100 experimental rats were randomly divided into 4 group:control group(A group),CsA group(B group),DC group(C group),imDC group(D group).(1)Control group(A group):Wistar rats accpeted liver transplantation without any treatment.(2)CsA group(B group):Wistar rats accpeted liver transplantation with 10mg/kg of CsA treatment.(3)DC group(C group)Wistar rats accept mature DC transfusion only.(4)ImDC group(D group):ImDC was transfused into Wistar rats in the amount of 1×10~6 days before operation.2 The level of ALT,AST,TBIL,IL-2,IFN-γ,IL-4andIL-10 were detected at certain time after transplantation.3 The percentage of CD4~+CD25~+ T cells were detected by flow cytometry.4 Pathologica studies of the acute rejection and the expression of FasL/Fas in the grafts were detected by HE and immunohistochemical staining.5 Western blot was used to detect Scurfin protein expression of CD4~+CD25~+ T cells.RESULTS:1 The median survival time(MST)of the liver allografts in CsA group and imDC group were significantly longer than control group and DCgroup(P＜0.05).2 The level of ALT,AST and TBIL in control group and DC group were rapid advanced but gradually recoveried in CsA group andimDC group.The level of IL-2 andIFN-γin control group and DCgroup were higher than CsA control group and imDC group(P＜0.01),The level of IL-4 and IL-10 in control group and DCgroup were lower than CsA group and imDC group at the same time(P＜0.01).3 The percentage of CD4~+CD25~+ T cells were higher than other three groups at day 7 and 10(P＜0.05).4 Histological examination,slightly or no rejection reaction were found in CsA group and imDC group.5 The scurfin protein expression of CD4~+CD25~+ T cells had the same result as the percentage of CD4~+CD25~+ T cells detected by flow cytometry.Conclusion:1 Application of onpufsed allopeptide syngeneic Recipient-Derived immature Dendritic Cells is an effective way to induce immune Hyporesponsivenes by blockading indirect recognition in rat liver transplantation model,survival span was signicant]y prolonged by its protective effect.2 The mechanism of immune hyporesponsivenes induced by immature dendritic cells transfusion might be involved in some aspects:T cell apoptosis,T cell anergy to allogeneic graft,immune deviation of TH1/TH2 cytokine net and inhibition of T lymphocytes responsiveness by Regulatory T cells.