The Experimental Research On The Suppressive Effect Of IDO To Acute Rejection Of Small Bowel Transplantation In Mice

The aim of this research project is to investigate the expression of indoleamine 2,3-dioxygenase(IDO) in murine myeloid and splenic dendritic cells (DC) and its effect to allogenic T lymphocyte proliferation. To design to pre-operatively intravenous infuse donor-derived DC with IDO high expression into allogenic recipient in order to determine its inhibition effect to acute rejection in mice segmental small bowel transplantation (SBT). Furthermore, combine medication consisting of Sinomenine and donor-derived DC with IDO high expression is used and evaluated for their anti-rejection effects.Part I The expression of IDO in murine DC and its effect to allogenic T lymphocyte proliferationObjective: To investigate the phenotype and IDO expression of murine myeloid and splenic DC, study its effect to allogenic T lymphocyte proliferation. Methods: DCs were isolated from murine bone marrow and spleen then cultured through method of separating and inducing. The phenotypes of DCs were determined by flowcytometry. The IDO expression level and activity of DCs with or without Interferon-y induction were investigated by RT-PCR, western blot and capillary electrophoresis. The alterations in stimulating ability to allogenic T lymphocyte proliferation through DCs with/without Interferon-y induction were observed by single MLC. Results: About l-2×10~7 myeloid DC and 3-5×10~6splenic DC were harvested from each mouse. Those cells showed typical dendrite-like appearance. No differences of MHClP, CD11C+, CD80+ and CD86+ were found between these two types of DCs(all P>0.05), meanwhile the CD8a+in mice splenic DC (15.46±0.85%) was significantly higher than that in myeloid DC (1.68±0.16%,P<0.01) . The expressions of IDO mRNA and protein in murine splenic and myeloid DC were similar, and correspondingly increased by IFN-y induction. The IDO secreting by splenic DC had the function of disintegrating tryptophan to kynurenine, and it was increasing with IFN-y induction, in contrast IDO from myeloid DC continually appeared very lower ability whatever with or without IFN-y induction. The stimulating allogenic T lymphocyte proliferation of splenic DC was significantly lower than that of myeloid DC and getting down more with IFN-y induction. This stimulating function of splenic DC could be enhanced to the equal level with myeloid DC by application of specific inhibitor 1-methyl-tryptophan (1-MT). Conclusion: Comparing with murine myeloid DC, differences are found as that the murine splenic DCs had about 15% CD8a+DC with ability of functional IDO expression which could be secreted by CD8a+DC. The high expression of functional IDO from splenic DC was due to IFN-y induction and make stimulating ability of splenic DC to allogenic T cells proliferation lower.Part II The establishment of segmental heterotopic small bowel transplantation model in miceObjective: To establish stable segmental heterotopic small bowel transplantation model in mice for advanced study in vivo. Methods: The establishment of SBT model by microsurgical procedure was divided to 2 stages—technical training stage (stage l)and experimental investigation stage(stage 2). Results: 108 operations were performed with 72.22% survival inthe stage 2 which was significantly higher than it in the stage 1(27.05%, PO.01). In the stage 2, although the failure of angioanastomosis was quite less than in the stage 1, the main reasons of recipient death clearly were poor perfusion and preserving of grafts. Comparing with the stage 1, all of time of graft cold and warm ischemia or donor and recipient operations in the stage 2 were considerably shorter(P<0.01). Conclusion: To ensure the quality of grafts and perfect angioanastomose is the key to operation success. Bowel perfusion prior to blood vessel and effective bleeding precaution should be most important.Part III The inhibition effect of DC high expressing functional IDO to acute rejection in mice SBTObjective: To study the effect and mechanism of donor-derived DC high expressing functional IDO to acute rejection in mice SBT, investigate the expression of IDO in grafts and its significance. Methods: Both of donor-derived splenic DC induced by IFN-y high expressing IDO (induced DC for short) and DC from the same source but without IFN-y induction low expressing IDO (DC for short) were preoperatively intravenous injected to allogenic mice, that were the subgroups land 2. For the control, subgroups 3, 4, and 5 were divided as the following that subgroup 3( isograft), subgroup 4(allograft) and subgroup 5( allograft with CsA treatment). The survivals of grafts and recipients were observed. On the post-operation day (POD)6, grafts were individually collected for pathological investigation, meanwhile mRNA expression levels of IL-2, IFN-y, IL-10 and IDO were assayed by RT-PCR, as well as IDO protein in grafts were determined by Western Blot method. Results: Comparing with subgroups 4 and 2, the graft survival of subgroup 1 was significantly longer (P<0.0\), its pathological features were downgrading companying with significantly lower levels of IL-2 and IFN-y mRNA expression (i><0.01), however its IL-10 , IDOmRNA expression and IDO protein levels were notably higher (PO.01). Furthermore in the comparison with subgroup 5, the shorter survival, higher level of IL-2 mRNA and lower IL-10 mRNA expression in grafts were found in subgroup l(all P<0.0l), but others. There were negative correlations between pathological grading of graft and its IDO expression or cytokines Th2 levels (PO.Ol), in contrast positively correlative with Thl cytokines (P<0.01). Conclusion: Pre-operative medication with donor-derived DC high expressing functional IDO may inhibit rejection in mice SBT. This inhibition seems to be relative with inducing IDO expression in graft and leading the deviation from Thl toTh2.Part IV The inhibiting effect to acute rejection in mice SBT by donor-derived DC high expressing functional IDOcombining SinomenineObjective: To study the inhibiting effect and its mechanism to acute rejection in mice SBT by combining medication of donor-derived DC high expressing functional IDO pre-operatively intravenous infusing and Sinomenine post-operative injection. Methods: The subgroups were set up as the followings: subgroup 1 (pre-operatively infuse donor-derived DC high expressing functional IDO combining post-operatively inject Sinomenine), subgroup 2 ( no treatment), subgroup 3 (use DC induced by IFN-y), subgroup 4 ( use Sinomenine only), and subgroup 5 (use CsA only). The survivals of grafts and recipients were observed. On the POD 6, grafts were collected and pathologically examined, meanwhile mRNA expression levels of IL-2, IFN-y, IL-10 and IDO were assayed by RT-PCR, as well as IDO protein in grafts were determined by Western Blot method. Results: Comparing with subgroup 2, although the longer survival of graft, lower pathological grading and IFN-y mRNA expression were found, no...