Study Of The Inhibition On The Rejection In Renal Transplantation For Rats By Treg-derived Exosome Induced By Oral Administrated RDP58

Background and Objective:Allograft rejection was the main reason of transplanted organ to lose their function andthe main obstacle to survive long-term which is the important tasks in the area oftransplantation. Though the rational use of all kinds of immunosuppressant had effectivelyreduced the organ rejection and increased the success rate of graft transplantation and keptgood functions in allograft long-term survival and function maintenance, it had it'sinsurmountable defect-suppressing the normal immune function.Oral tolerance is the acting state of the body's immune system to a large number ofmicroorganism and food; which is the acquired mechanism of induce tolerance to theforeign antigen (food antigen, intestinal bacteria symbiosis antigen). Among them,regulatory T cells (regulatory T cells, Treg) in the mesenteric lymph node of the lymphaticsystem played a vital role in above tolerance.The CD4+CD25+Treg had important immune adjustment function and had thenegative regulation effect in the activation and proliferation of effect T cells, At the sametime, which played a very important role in induce immune tolerance and maintain thetolerance state after transplantation. Effective use of itself CD4+CD25+Treg can avoidadverse effect of immunosuppressive agents in suppress the rejection in allograft and therecipients. As known, exosome is secreted by a variety of living cells in the body in themanner of exocytosis; and different cell sources of exosome had different immune function,such as DC sources of exosome and B cells sources of exosome had many MHC IImolecules and stimulus molecules (CD86, CD80), which was the necessary of T-cellactivation; The exosomes secreted by nasopharyngeal carcinoma had latent membraneprotein gene1(LMP1), which could restrain T cell activation effect; exosomes secreted bysome cancer could abnormality express Fas ligand (FasL) and induce activated induced cell death (AICD). It had been confirmed that B cells, T cells, DC cells, mast cells and a widevariety of tumor cells all can secrete exosome. Then can CD4+CD25+Treg cells secreteexosome and accomplish the far-end regulation by the secreted exosome? To prove this idea,we collected the intestinal CD4+CD25+Treg cells induced by oral administration of RDP58peptide conjugated to cholera toxin B subunit (CTB) and collected the CD4+CD25+regulatory T cells-derived exosomes.Materials and Methods:This experiment was to collect the RDP58-CTB induced specific CD4+CD25+regulatory T cells-derived exosomes and establish the rat transplantation model to observethe biology function of exosomes in vivo and in vitro after adoptive transfer and its proteincomposition by SDS-Page and High Performance Liquid Chromatography-CHIP-MassSpectrometer (HPLC-CHIP-MS):1. The synthetic of RDP58peptide and the test of the molecular weight and purity;2. The conjugated of RDP58to CTB used SPDP as cross linking agent and detectionthe purity of RDP58-CTB by HPLC;3. We established rat kidney transplantation models and observed the effect oftolerance inducted by oral administration of RDP58-CTB and observed the change offunction by derivatizing the structure of RDP58peptide;4. Induced mesenteric specificity CD4+CD25+Treg by oral administration ofRDP58-CTB;5. We collected the mesenteric lymph nodes and sorted CD4+CD25+Treg byFluorescence-activated cell sorting (FACS) technology, then we cultured theCD4+CD25+Treg and collected the exosome from the supernatant;6. After established rats kidney transplantation models, we adoptive transferredCD4+CD25+Treg-derived exosomes and observed its biology function;7. The analysis of exosome protein by SDS-Page and HPLC-CHIP-MS.Results:1. The purity of RDP58peptide and derivative RDP58peptide which was synthesizedwas both more than95%; and with the help of SPDP as cross linking agent, we completedRDP58derivative peptide conjugated to CTB;2. With the help of prolene as stent, we established the rats kidney transplantation and effectively improved the rate of success and reduced ischemia times;3. Oral administration of RDP58conjugated to CTB could effectively improve theactivity of HO-1in vivo,suppress the proliferation of T cells and significantly improve thesurvival and histopathology of allograft kidney tissue relative to the oral administration ofRDP58alone, which got the same treatment effect as the intraperitoneal injection ofRDP58;4. we used ultracentrifugation and density gradient centrifugation to isolate and purifyexosomes, which had the diameters of30-100nm, as observed by TEM,and the densities of1.13to1.21g/ml (floated on30%sucrose/D2O)ï¼›5. Exosome adoptively transferred into the rats could significantly prolong the survivalof transplanted kidney in vivo and suppress the proliferation of T cells in vitro;We preliminarily observed the quantity and the kinds of proteins by runningSDS-polyacrylamide gel (SDS-Page) electrophoresis, and further analyzed it byHPLC-CHIP-MS and found that there were dozens of protein, including cap adenylylcyclase-associated protein1, ldha L-lactate dehydrogenase A chain and so on.Conclusions:1. RDP58peptide conjugated to CTB could effectively enhance the antigenimmunogenicity, significantly prolong the survival of graft and increased the activity ofHO-1in vivo; and get the same effect as intraperitoneal injection of RDP58, however oraladministration of peptides was more safe and convenience;2. CD4+CD25+Treg cells could secrete exosomes which could significantly improvethe survival of allograft and the pathological changes and might be one of the far-endregulation mechanisms of CD4+CD25+Treg cells;3. It was confirmed that exosome contained more than10proteins by the analysis ofSDS-Page and HPLC-CHIP-MS.