| Part 1:Establishment of heterotopic small bowel transplantation model in miceObjectives:To establish a stable model of heterotopic small bowel transplantation (SBT) in mice.Methods:The operation was made as following:the donor portal vein was anastomosed end by side with recipient IVC, the donor SMA with aorta patch was anastomosed end by side with recipient abdominal aorta, proximal end of the intestine was ligated, the distant end of the intestine was anastomosed end by side with recipient intestine. We choose C57BL/6 mice as donor and the BALB/c mice as recipient. The mice were fasted for 3 days after the operation, no drink limited,2ml 5% glucose saline was injected two times everyday. There is no antibiotic and immunosuppressor.Results:50 cases were made in the early stage of modeling with 9 cases completed the operation, while other 41 cases died during the transplantation. The causes of death were:artery anastomotic thrombosis in 20 cases, massive hemorrhage in 8 cases, vein anastomotic thrombosis in 5 cases, anastomosis stenosis in 3 cases, anesthetic accident in 3 cases, abdominal infection in 1 case and with unknown reason in 1 case. In the stable stage of modeling 60 cases were made with donor harvest time 35-42min, warm ischemic duration 0.5min, donor preparation time 3-4min, cold storage of donor duration 25-34min, recipient operation time 77-91min, the abdominal aorta and IVC blocked time 33-45min, vein anastomosis time 8-12min, artery anastomosis time 13-19min, the average blood loss 0.2ml.24h survival rate 80%(48/60), POD5 survival rate 70%(42/60); 18 cases died during the operation or within POD5 by: artery anastomotic thrombosis in 8 cases, anastomosis stenosis in 4 cases, massive hemorrhage in 4cases, abdominal infection in 1 case and with unknown reason in 1 case.Conclusions:1. The modified operation can be used to establish a stable model of the mouse heterotopic small bowel transplantation for the advanced research in the future;2. It is difficult to perform small bowel transplantation in mice, and the sticking points to success are high quality donor, refined anastomosis techniques and good liquid management. Part 2:Study of the rejection after small bowel transplantation in miceObjectives:To explore the roles of immunocytes and cytokines in the rejection after small bowel transplantation in mice, elucidate the probable mechanism of the effects, and to find the good indicators reflecting the rejection.Methods:We choose C57BL/6 or BALB/c mice as donors and BALB/c mice as recipients, and all mice were male. The mice were randomly divided into 4 groups, n=6:Group A, BALB/c→BALB/c, POD11; Group B, C57BL/6→BALB/c, POD3; Group C, C57BL/6→BALB/c, POD7; Group D, C57BL/6→BALB/c, POD11. The transplantation was made as we described in part 1, and the mice were sacrificed on the scheduled day. Spleens were weighed and counted the cells; the concentrations of IL-10, IFN-y and IL-17 in serum were measured by ELISA; grafts were stained by Hematoxylin-Eosin for pathological observation; and we use flow cytometry to test the subset of the immunocytes in spleen cells and grafts’infiltrating cells.Results:Pathological findings of grafts show:no rejection in group A, mild rejection in group B, moderate rejection in group C and severe rejection in group D; there were significant differences in all groups of IL-10 and IL-17 serum level(P<0.05), IL-10 level decreased in turn in group A,B,C and D, IL-17 level increased in turn in group A,B,C and D; for the serum level of IFN-γ, there were no significant differences between group A and B (P>0.05), and significant differences in group B,C and D(P<0.05), it increased in turn in group B,C and D; there were significant differences in all groups of spleen weight and cell numbers(P<0.05), they increased in turn in group A,B,C and D; for the ratios of T cell and B cell in spleen cells, there was no significant difference between group A and B (P>0.05), and significant differences in group B,C and D(P<0.05), they decreased in turn in group B,C and D; for the ratios of neutrophils in spleen cells, there were significant differences in all groups(P<0.05), they increased in turn in group A,B,C and D; for the ratios of CD4+T cells in spleen T cells, there were no significant differences between group A and B, group C and D (P>0.05), compare to group B, it significant decreased in group C(P<0.05); for the ratios·of CD8+ T cells in spleen T cells, there were no significant differences between group A and B, group C and D (P>0.05), compare to group B, it significant increased in group C(P<0.05); for the ratios of CD4-CD8- T cells in spleen T cells, there was no significant difference between group A and B (P>0.05), and significant differences in group B,C and D(P<0.05), it increased in turn in group B,C and D; for the ratios of Thl in spleen CD4+ T cells and Tcl in spleen CD8+ T cells, there were no significant differences between group A and B (P>0.05), and significant differences in groups B,C and D(P<0.05), they increased in turn in group B,C and D; for the ratios of Th2 and Th17 in spleen CD4+ T cells, there were significant differences in all groups(P<0.05), Th2 decreased in turn in group A,B,C and D, but Th17 increased in turn in group A,B,C and D; for the ratios of T cells in graft infiltrating cells, there was no significant difference between group C and D (P>0.05), and significant differences in group A, B and C(P<0.05), it decreased in turn in group A, B and C; for the ratios of B cells and neutrophils in graft infiltrating cells, there were significant differences in all groups(P<0.05), B cells decreased in turn in group A, B, C and D, but neutrophils increased in turn in group A,B,C and D; for the ratios of CD4+ T cells and CD8+ T cells in graft infiltrating T cells, there were significant differences in all groups(P<0.05), CD4+ T cells decreased in turn in group A, B, C and D, but CD8+ T cells increased in turn in group A, B, C and D; for the ratios of CD4-CD8- T cells in graft infiltrating T cells, there were no significant differences in all groups(P>0.05); for the ratios of Thl and Thl7 cells in graft infiltrating CD4+ T cells and Tcl cells in graft infiltrating CD8+ T cells, there were significant differences in all groups(P<0.05), they increased in turn in group A, B, C and D; for the ratios of Th2 cells in graft infiltrating CD4+ T cells, there was no significant difference between group A and B (P>0.05), and significant differences in groups B,C and D(P<0.05), it decreased in turn in group B, C and D. Conclusions:1. Rejection after small bowel transplantation is a complex process involves a variety of immunocytes and cytokines, in which T cells play a key role;2. Th1, Th17 and Tcl cells are the very important cells in the process of rejection, the ratios of these cells and their cytokines IFN-γand IL-17 can be used as indicators of rejection;3. Th2 cells and their cytokines IL-10 can reduce the rejection, they can induce tolerance.Part 3:The study of roles of TLRs in the rejection and tolerance after small bowel transplantation in mice and their mechanismsObjectives:To explore the roles and probable mechanisms of TLRs in the rejection after small bowel transplantation in mice, and to study the possibility and probable mechanisms of tolerance after small bowel transplantation in mice by blocking the TLRs signaling pathways.Methods:We choose C57BL/6 mice as donors and wild type or MyD88-/- BALB/c mice as recipients, and all mice were male. The mice were randomly divided into 4 groups:Group A, C57BL/6→BALB/c, POD7; Group B, C57BL/6→MyD88-/-BALB/c, POD7; Group C, C57BL/6→BALB/c, POD11; Group D, C57BL/6→MyD88-/-BALB/c, POD11. The transplantation was made as we described in part 1, and the mice were sacrificed on the scheduled day. Each group was repeated 7 times,1 was used for immunohistochemistry staining, others experiments were repeated for 6 times (n=6). Spleens were weighed and counted the cells; the concentrations of IL-10, IFN-y and IL-17 in serum were measured by ELISA; grafts were stained by Hematoxylin-Eosin for pathological observation; and we use flow cytometry to test the subset of the immunocytes in spleen cells and grafts’infiltrating cells; and the spleen cells were used for MLR.Results:Pathological findings of grafts show:moderate rejection in group A, mild rejection in group B, severe rejection in group C and mild-moderate rejection in group D. The IL-10 level in serum, ratios of Treg and CD4+T cells in spleen and grafts infiltrating T cells, ratios of Th2 cells in spleen and grafts infiltrating CD4+T cells and the numbers of Treg in spleen, compare to group A, they were significantly increased in group B (P<0.05); and compare to group C, they were significantly increased in group D (P<0.05). The IFN-γand IL-17 level in serum, spleen weight and cell numbers, ratios of CD8+T cells in grafts infiltrating T cells, ratios of Thl, Th17 cells in spleen and grafts infiltrating CD4+T cells, ratios of Tcl cells in spleen and grafts infiltrating CD8+ T cells and the stimulate index in MLR, compare to group A, they were significantly decreased in group B (P<0.05); and compare to group C, they were significantly decreased in group D (P<0.05). For the ratios of CD4-CD8-T cells in grafts infiltrating T cells, there were no significant differences between group A and B, group C and D(P>0.05).Conclusions:1. TLRs play an important role in the development of rejection after small bowel transplantation, the tolerance can be induced by blocking the TLRs signaling pathways;2. The CD4+Foxp3+ Treg cells can induce and keep the tolerance after small bowel transplantation, and the mechanisms may be related with IL-10;3. TLRs may be affected by regulation of Treg in the rejection after small bowel transplantation, and blocking the TLRs signaling pathways may increase the number of Treg cells which induce the tolerance. |
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