The Experimental Research On Prophylaxis Of Chronic Rejection In Rat Small Bowel Transplantation With Chinese Herbs

SectionⅠThe Establishment and Characterization of the Rat Small BowelTransplantation ModelObjective: To establish the rat small bowel transplantation model and investigatethe mechanism of rejection. Methods: Isogeneic and allogeneic small boweltransplantation were performed in rats with microsurgical technology. Recipients weredivided into three groups: isogeneic control, allogeneic control, and allogeneictransplantation with CsA treatment (CsA group). Graft tissue were harvested on POD7,POD14, POD28 and POD60 and routine pathological examination was carried out.To monitor the acute rejection, the mRNA of IL-4 and IFN-γ, were quantified byreal-time quantitative RT-PCR. The infiltration of macrophages was determined bydetecting CD68 with immunohistochemical staining. In order to investigate therelation of TGFβ1 and PDGF with graft rejection, mRNA transcription and proteinexpression of TGFβ1 and PDGF were measured by real-time quantitative RT-PCR andWestern blot analysis. Immunohistochemical staining was also performed to detect thelocation of TGFβ1 and PDGF expression. Results: (1)All recipients in allogeneiccontrol group were died within 11 days after transplantation. (2)There were significantincreasing of IL-4 and IFN-γmRNA transcription in CsA group in POD7 comparedwith isogeneic group, but no significant differences were detected in POD14, POD28and POD60. (3)The chronic rejection scores of CsA group in POD60 were higher thanthat in isogeneic group. (4)CD68 expression in CsA group increasing significantly inPOD60. (5)Both the mRNA and protein expression of TGFβ1 and PDGF wereincreased significantly. The immunohistochemical results showed a high expression ofTGFβ1 and PDGF in the matrix. Conclusion: (1)Acute rejection occurred in the early stage of CsA group. (2)There was extensive infusion of macrophages in the graftof CsA group in POD60. (3)Chronic rejection was occurred in CsA group in POD60,with the increased levels of TGFβ1 and PDGF. (4)The chronic rejection model ofallogeneic small bowel transplantation could be established successfully within 60days after operation. The expression of CD68, TGFβ1 and PDGF were significantlyincreased, which could be used to determine chronic rejection.SectionⅡThe Research on Prophylaxis of Chronic Rejection with Single Chinese Herbin Rat Small Bowel TransplantationObjectives To demonstrate the prophylaxis of chronic rejection with single Chineseherb and select the best one. Methods Isogeneic and Allogeneic small boweltransplantation were performed in rats with microsurgical technology. After CsAtreatment of allogeneic recipients for one month, all experimental recipients weretreated with Tripterygii Wilfordii, Tetrandrine and Sinomenine, respectively. At thesame time, allogeneic control and isogeneic control were designed. Graft tissue wereharvested in POD7, POD14, POD28 and POD60. Routine pathological examinationwas carried out. The infiltration of macrophages was determined by detecting CD68with immunohistochemical staining. The mRNA transcription and expression ofTGFβ1 and PDGF were measured by real-time quantitative RT-PCR, Western blotanalysis and Immunohistochemical staining. T cell subsets in peripheral blood andgraft in the end of research were analyzed by flow cytometry measure. Results (1)The pathological scores of Tetrandrine group, Sinomenine group and allogeneiccontrol in POD90 increased significantly than the other groups of the same stage. (2)CD68 detected by immunohistochemical staining showed higher expression in thegroups of Sinomenine, Tetrandrine and allogeneic control in POD90. (3)There werealso significant increase of mRNA transcription and expression of TGFβ1 and PDGF in the groups of Sinomenine, Tetrandrine and allogeneic control in POD60 andPOD90. (4) Flow cytometry measure showed decrease of CD4+/CDS+ in peripheralblood in Tripterygii Wilfordii group and Tetrandrine group, and CD4+/CDS+ in thegrafts of allogeneic control decreased either. (5)AST were increased meaningfully inTetrandrine and Sinomenine groups. Conclusion (1)Serious damages andmacrophages infiltration were existed in Tetrandrine group, Sinomenine group andallogeneic control. (2)TGFβ1 and PDGF were significantly increased in the groups ofSinomenine, Tetrandrine and allogeneic control in POD60 and POD90. (3) Thedecrease of CD4+/CD8+ in peripheral blood and graft in Tripterygii Wilfordii,Tetrandrine group and allogeneic control showed the effect of immunoimpression ofthese herbs, and the damages of the immune function in grafts of allogeneic group. (4)Tetrandrine and Sinolnenine showed side-effects to liver function. (5)Compared withthe other herbs, Tripterygii Wilfordii could be the best one to control the cytokines ofTGFβ1 and PDGF, and thus prophylaxes the chronic rejection.SectionⅢThe Research on Prophylaxis of Chronic Rejection with Compound Chinese Herbfollowing Small Bowel TransplantationObjectives To demonstrate the prophylaxis of chronic rejection with compoundChinese herbs and choose the effective mixtures. Methods Isogeneic and allogeneicsmall bowel transplantation were performed in rats with microsurgical technology.After CsA treatment of allogeneic recipients for one month, all experimentalrecipients were treated with Tripterygii Wilfordii-Radix Astragali mixture, TripterygiiWilfordii-tanshinone mixture and Tripterygii Wilfordii- Radix Astragali- tanshinonemixture, respectively. At the same time, allogeneic control and isogeneic control weredesigned. Graft tissue were harvested in POD7, POD14, POD28 and POD60.Routine pathological examination was carried out. CD68 was detected by immunohistochemical staining. The mRNA transcription and protein expression ofTGFβ1 and PDGF were measured by real-time quantitative RT-PCR, Western blotanalysis and Immunohistochemical staining. The T cell subsets in peripheral bloodand graft in the end of research were analyzed by flow cytometry measure. Results(1)The pathological scores of Tanshinone group, Radix Astragali-tanshinone group,and altogeneic control in POD90 increased significantly than the other groups of thesame stage. (2)Compared with the other groups, CD68 expression in allogeneiccontrol increased significantly in POD90. (3)There were significant increase ofmRNA transcription and expression of TGFβ1 and PDGF in POD60 and POD90. Thecomparison in POD60 showed the higher level of mRNA transcription and expressionof TGFβ1 and PDGF in allogeneic control, and in POD90, the groups of allogeneiccontrol, Tanshinone, and Radix Astragali-tanshinone showed significant increasethan the other groups. (4)The CD4+/CDS+ in the grafts of allogeneic control werelower than the other groups, and no difference of CD4+/CD8+ in peripheral blooddetected. Conelusion (1)Serious damages were existed in Tanshinone group, RadixAstragali- tanshinone group, and allogeneic control in POD90. (2)There wereintensive infiltration of macrophages in group of allogeneic control. (3)TGFβ1 andPDGF were significantly increased in allogeneic control in POD60, and groups ofallogeneic control, Tanshinone, and Radix Astragali-tanshinone in POD90. (4) therewas damages of the immune function exist in grafts of allogeneic group. (5)Comparedwith the other groups, Tripterygii Wilfordii- tanshinone mixture could be the bestcompound inhibiting the expression of TGF-β1 and PDGF , and alleviating theinfusion of macrophages, which could be used for prophylaxis of chronic rejection.