The Empirical Study Of ImDC Modified By TGF-β1 To Induce Immune Tolerance In Rat Heterotopic Small Bowel Transplantation

Organ transplantation, one of hot medical fields ,has gained great achievement as an alternative for the treatment of some diseases of terminal stage,but rejections following organ transplantation have still been puzzling tough problems. Immunodepressant plays a pivotal role in preventing and curing graft rejections in both reseach and clinical practice,but it can cause side effects such as hypertension,hepar and renal toxicity,severe opportunistic infection,malignant tumor and even malfunction of grafts,etc.Nowadays,hot and tough problems in organ transplantation field are how to induce immune tolerance of grafts.for the past few years,along with lucubrating APC, especially DC,it's well acknowledged that DCs were the most effective APC-only DC can activate incipient Tcells.Consequently,regulating paths of DC allergization may induce immune tolerance of grafts and prolong survival time of grafts.Contemporily,new immune factor TGF-pi and ripening techniques of DC culture in vitro make it possible that gene-modified DC induces immune tolerance of grafts. TGF-pi is one of powerful immune suppressor factors,which participates in interaction of immune cells,influences proliferation and differentiation of thymocytes and NK cells,interferes production and transformation of IG from B cells,remotely influences IL-1,IL-2 and IFN-γ,inhibits MHC and CM expression of DCs,therebydegrades DCs antigen presentation function prevents mature and differentiation of DCs ,and finally forms immune tolerance of grafts.Part OneConstruction of adenovirus vector recombinated by TGF-β1 and identification of fuction of transfected DCsObjectives:To construct and identify the adenovirus vector recombinated by TGF-β1,transfect TGF-P1+ virus into DCs in vitro,then detect bionomics of the DCs and observe the effects.Methods: Prepare of rat HBSC,select CD34+ cells by immunomagnetic beads and cultivate them with GM-CSF of different dosis,collect cells of different time for immunophenotype identification ,ultrastructure observation and MLR to observe the levels of T cells proliferation induced;Magnify TGF-β1 by RT-PCR and identify positive clones;Construct and identify shuttle vectors pAdTrack-CMV recombinated by TGF-β1,then collect and pack recombinated adenovirus vectors and assess them by virus titer;transfer TGF-pi gene into rat DCs in vitro culture,detect expression of TGF-β1 and bionomics of the transfered DC by Western blot and rat vascular endothelial cell GI experiment,observe how the transfered DC influences T cells proliferation by MLR.Results:DCs cultivated with small dose of GM-CSF could represent features of immature DCs, that cell surface with low-expressed CD80,CD86 and MHC-II ,and high-expressed CD11c and CD40, and weak ability to stimulate native T cells to proliferate. But DCs cultivated with large dose of GM-CSF could represent features of DCs, that cell surface with high-expressed CD80,CD86 and MHC- II ,and strong ability to stimulate native T cells to proliferate.And we succeeded in amplifying TGF-pi and subcloning its gene frags into shuttl vector pAdTrack-CMV,then transfect it into HEK293 cells , collect AD virusand obtain TGF- β1+ AD virus(titre 10¹²PFU/ml).After that, we measured the levels of GFP by fluorescence microscope, which shew that virue was packed successfully and that transfected TGF-β1 DC could secrete TGF-β1 and restrain increasing of vascular endothelial cells and T cells. Conclusions:Method of immunomagnetic beads could obviously increase pick-up rate of DCs.In cultivations of different dose GM-CSF and on different time points,we could obtain DCs of different maturity.Applying with small dose of GM-CSF could induce to generate immature DCs with typical function.Applying with RT-PCR could amplify TGF-β1 gene from rat small bowel and construct shuttle vector of gene recombination.By homologous recombination and pack of recombinated AD virus and measure of virus titre,we could obtain high-expressed target genes.And at the same time,virus titre was wholly in line with requirements of next experiment. Transfecting TGF-β1+ adenovirus vector into rat DCs , TGF-β1 gene could express stably in DCs and the DCs possessed stable bionomics. Part TwoThe empirical study of imDC transfected by TGF-β1- recombinatedadenovirus vector to induce immune tolerancein ratheterotopic small bowel transplantationObjectives: To establish rat small intestine transplantation model of graft rejective reaction and investigate mechanisms of imDC transfected by TGF-β1 -recombinated adenovirus vector to induce immune tolerance by the regulation of regulatory T cells and apoptosis in rat heterotopic SBT. Methods: Heterotopic small bowel transplantations were performed from F344/N donors to BN recipients.The recipients were divided into four groups(n=24): isograftgroup(BN-BN),allograftgroup,TGF- β1 allograft group,FK506-TGF-β1 allograft group.In TGF-β1 allograft group and FK506-TGF-β1 allograft group, recipients were given TGF-β1-transfected 10⁷ imDCs intramuscularly daily five days Before OP . In FK506-TGF-β1allograft group, recipients were given 0.2 mg/kg FK506 intramuscularly daily after OD. On POD 3,5, and 7,the entire graft jejunum and peripheral blood from 6 recipients of each group were taken for immunochemistry to assess the levels of CD4, CD8, CD25(IL-2R) and IL-4, for TURNEL to observe apoptosis in grafts and for transmission electron microscope to observe grafts ultramicrostructure respectively. Remnant rats of each group were for analysis of median survival. Recepients died within 72h were considered for technique failure and not for statistics.Results: In allograft group, typical mild,moderate and severe acute rejection occurred Isograft group rejection on POD 3 ,5 and 7.Both CD4 and CD25 were high-expressed, while CD8 and IL-4 were low-expressed. The levels of Bcl-2 were degraded, while the levels of Bax were a bit elevated and the levels of Bcl-2/Bax were significantly elevated.massive apoptosis were observed on POD 5 by TURNEL and electron microscope. Rat median survival time was 9.35±2.71 days; In isograft group, no acute rejection was found.The levels of CD4, CD25, CD8, IL-4, Bcl-2 and Bax were not obviously changed. Apoptosis could be observed by chance. Rat median survival time was 28.30±3.16 days; In TGF-(31 group, mild acute rejection still occurred with pathohistology signs on POD 7. The levels of Bcl-2 and Bcl-2/Bax were significantly elevated, while the level of Bax were degraded. Among lymphocytes infiltrating into bowel graftS, CD4+, CD8+ and CD25+ cells were obviously degraded, a little a- poptosis could be observed on POD 7 by TURNEL and electron microscope. Rat median survival time was 16.32±5.41 days. In FasL-TGF-β1 group, histopathology signs of all bowel grafts shew no acute rejection. Among lymphocytes infiltrating into bowel graft, CD4+, CD8+ and CD25+ cells were significantly degraded. The levels of IL-4 and Bcl-2/Bax were significantly...