Prolongation Of Rat Intestinal Recipients' Survival By Administration Of Exosomes Derived From Donor Immature Dendritic Cells

Prolongation of Rat Intestinal Recipients'Survival by Administration of exosomes derived from Donor Immature Dendritic CellsChapter 1 Establishment of Intestinal Transplantation Model in RatsObjective To investigate the surgical procedures and to establish stable intestinal transplantation model in rats.Methods Segmental heterotopic intestinal transplantation was performed from donor F344 rats to recipient Wistar rats. A segment of intestine about 10cm with vessels was harvested from donor F344 rats. The vascular reconstruction was as follows: donor superior mesenteric artery was anastomosed to recipient infrarenal aorta and donor portal vein to recipient inferior vena cave. The proximal end of graft was ligatured and the distal end was exteriorized as a stoma on the abdominal wall.Results A total of 56 rats underwent intestinal transplantation with 50 rats survived more than 3 days(89.3%). The donor operation time was 37.3±1.6min, and the recipient's 57.3±4.1 min. Of the operation time, the anatomosis of artery toke 10.4±1.6 min, and that of vein8.9±0.7 min. The cold ischemia time of graft was 21.9±2.1 min and the warm ischemia time 19.3±2.0min. The survival of recipients without any preconditioning varied from 5 days to 8 days, with an average of 7.0±0.6 days. Conclusion This intestinal transplantation model by us is stable, reliable and useful for the further study of intestinal transplantation. And this makes a foundation of the followed experiments.KChapter 2 The Isolation and Identification of Exosomes from Rat Bone Marrow Derived Immature Dendritic Cells in vitroObjective To study the effective method for in vitro isolation and identification of exosomes from rat bone marrow-derived immature dendritic cells (imDC).Methods Bone marrow progenitor cells were obtained from F344 rat femoral and tibial bones and then cultured in the culture medium with 5ng/ml GM-CSF and 5ng/mlIL-4. On every other day, half of the medium was removed and equal volume of fresh medium wasadded. On day 6, non-adherent and loosely adherent cells were harvested and identified as imDC by flow cytometry, electronic microscope and ELISA. Exosomes from imDC culture supernatants were isolated by different ultracentrifugation and purified by ultrafiltration. To identify the characteristics,the exosomes from imDC were analyzed by flow cytometry ,electronic microscope and Mixed Lymphocyte Reaction (MLR).Results On day 6 of culture, imDC were harvested and identified. Morphological analysis by electronic microscope showed the typical propery of imDC. Surface phenotype assay by flow cytometry indicated the low to moderate expression of MHC classⅡ, and low expression of CD80 and CD86 of imDC. The concentrations of IL-12p70 detected by ELISA were significantly lower than those of mature DC(mDC) in the supernatant. The results demonstrated the DC generated from bone marrow progenitor cells was imDC. Exosomes from imDC culture supernatants were isolated by different ultracentrifugation and purified by ultrafiltration. Analysis of exosomes from imDC(imDex) by electron microscopy showed these particles were similar saucer-shaped vesicles(50-100nm) with clear bi-lipid membrane. After absorbed onto latex,phenotype of imDex was detected by flow cytometry. Results indicated that imDex expressd high MHC classⅡmolecules and low or undetectable CD80, CD86 and CD40 , which suggested they might bear the function of immune regulation. MLR meant that imDex induced lower T-cell proliferation than exosomes from mature DC(mDex) in the present of imDC.Conclusion It is an efficient and feasible method in this study of isolation exosomes from rat bone marrow-derived imDC in vitro. And this makes a foundation of the basic study of imDex.Chapter 3 Administration of exosomes from imDC prolonged the recipients'survival of intestinal transplantation in ratsObjective To investigate the effects and the mechanisms of administration of imDex could prolong the recipients'survival of intestinal transplantation in rats. Methods Segmental heterotopic intestinal transplantation was performed from donor F344 rats to recipient Wistar rats. Firstly,different doses of donor-imDex were injected into recipient rats via the vein. Recipient rats were divided into five groups according to the preconditionings that recipients received seven days before transplantation. Group A(n=6), in which recipients were injected with 1ml Normal Saline; Group B(n=6), in which recipients were injected with 1μg donor-imDex; Group C(n=6), in which recipients were injected with 10μg donor-imDex ; Group D(n=6), in which recipients were injected with 20μg donor-imDex and Group E(n=6), in which recipients were injected with 50μg donor-imDex. After intestinal transplantation, the survival of each groups were evaluated to gain the best suitable dosage of donor-imDex, which significantly prolonged the recipients'survival. According to the best suitable dosage of donor-imDex, rats were divied into two groups and the intestinal transplantation were repeated. No-treatment groups(n=6),in which recipients were injected with 1ml Normal Saline. The best suitable dosage of donor-imDex treated groups(n=6),in which recipients were injected with 1ml the best suitable dosage of donor-imDex. To investigate the effects of donor-imDex prolonged the recipients'survival, the cytokines including IL-2, IFN-γ, TNF-αand TGF-βin recipients'serum were test by ELISA. Mixed lymphocyte reaction was adopted to determine the ability of recipients'spleen T cell proliferation stimulated by donor spleen cells, and the cytokines of IL-10 and IFN-γin culture supernatants were also tested by ELISA. To further explore the mechanisms of imDex induction immune tolerance,the spleen CD4+CD25+T cell were analyzed by flow cytometry and the Foxp3mRNA of spleen T cells were detected by real-time PCR. And histologic examination of graft intestine was evaluated at the same time.Result: To test the tolerance-induced effects of imDex, we pretreated Wistar recipients with different doses (50, 20, 10 and 1μg of donor-type imDex) injected seven days before transplantation in the rat model of intestinal allotransplantation. Treatment with 10μg of donor-type imDex resulted in prolonged survival of allografts compared with those of the no-treatment groups (9.8±1.2 vs. 7.0±0.6 days, P<0.05, n=6), This effect increased with increasing doses of imDex, because pretreatment with 20μg imDex also significantly prolonged allograft survival, with a mean value of 14.5±1.0 days (compared with the no-treatment group, P <0.05; compared with the 10-μg group, P <0.05, n=6). However, overdose with imDex resulted in remarkably decreased allograft survival, whereas 50μg of imDex resulted in a mean value of 7.8±1.2 days (compared with the no-treatment group, n=6). We also tested the effect of 1μg of donor-derived exosomes, and we found no prolongation of allograft survival (7.3±1.0 days, compared with the no-treatment group, n=6). These results indicated that pretreatment of the recipient with a low dose (20μg) of donor-type imDex can modulate the acute intestinal allograft rejection. We next determined whether the effect of the imDex was donor-specific by injecting 20μg of recipient-imDex; this treatment had no significant effect on allograft survival (7.5±1.0 days, compared with the no-treatment group, n=6). For the purpose of histological examination, the intestinal grafts were evaluated on 5th day after transplantation. Although untreated recipient showed acute rejection with blunted villi, the number of goblet cells was decreased and a large amount of inflammatory cell infiltration occurred in lamina propria mucosa, the grafts treated with 20μg of donor-type imDex demonstrated mild lymphocyte infiltration and blunting of villi with edema. Administration of 20μg of donor-type imDex also decreased the levels of Pro-inflammatory Cytokines (IL-2,IFN-γand TNF-α),and increased the level of antio-inflammatory Cytokines TGF-βin Recipient Serum.To determine the immunoregulatory effect of imDex on allograft prolongation, the cellular response after transplantation was analyzed. Anti-donor T cells proliferative responses were assessed using purified splenic T-lymphocytes from 20μg imDex-treated animals and no-treatment animal at day 5 after transplantation. Total T-lymphocyte splenocytes (2.5×106 cells/mL) from the allograft recipients (responder cells) were co-cultured with donor splenocytes treated with mitomycin C (stimulator cells; 5×106 cells/mL). Cell proliferation was quantified by incubating the cells during the last 18 h of cultures with 1μCi 3Hthymidine. T-lymphocytes from 20μg imDex -treated rats proliferated less than cells from no-treatment recipients in response to donor splenocytes. This difference was also observed in the supernatant cytokine levels of IL-10 and IFN-γ. The concentrations of IFN-γwere significantly decreased, but IL-10 was greatly promoted among recipients infused with 20μg imDex. These data indicated that administration of imDex to allorecipients can inhibite anti-donor T-cell responses in vivo.The increase in TGF-βproduction in Recipient Serum prompted us to assess the capacity of imDex to induce Treg cells. To this end, the splenic T-cells of 20μg imDex-treated or no-treatment rats were collected seven days after transplantation. splenic CD4+CD25+ T-cells improved in 20-μg imDex-treated recipients in contrast with those of non-treated recipients. In addition, the Foxp3 mRNA expression demonstrated the same results.Conclusion Administration of imDex could prolong the survival time of recipients and alleviate the graft rejection. The up-regulated of CD4+CD25+Foxp3+ T cell may be one of the underlying mechanisms of induction immune tolerance by imDex.