| Background With the development of microsurgical technique and immune drugs, reconstruction using Vascularized Composite Allotransplant has brought new hope for patients with severe trauma, However, the postoperative complications of acute rejection is still the main challenge. Currently, the combination of a high dose of steroid and immunosuppressants is the mainstream treatment for acute rejection, but the associated opportunistic infection, tumor, diabetes and other side effects restricted the clinical application of.the treatment of acute rejection but the opportunistic infection(CMV, sepsis, cancer, diabetes and other side effects),restricted the allograft composite transplantation clinical promotion. In recent years, the induction of immune tolerance through cell-based therapy, namely donor specific transfusion(DST), in combination with low dose of immunosuppressant has demonstrated to be a prospective approach in a variety of animal models and human organ transplantation. The underlying mechanisms of DST include clone incompetence, activation induced cell death(AICD), and other immune regulation. Among these mechanisms, the IL-2 mediated AICD and induction of Tregs play an important part in the peripheral tolerance.The centrial issue of acute allogeneic rejection is the activation of immune effector cells. Whether DST would be able to regulate the host immune system in the early phase after allotransplantation so as to inhibit the effector cell activation and decrease the extent of rejection is a research interest of the field of VCA and, therefore, the focus of this thesis. In this study, the effects of intravenous infusion of donor splenocytes on the acute rejection of allogeneic skin grafts was investigated in a immunosuppressant-reduction induced acute rejection mouse model. The reduction of rejection extent and immunosuppressant dosage was evaluated. This study provides evidence for evaluation on the applicability of cell transfusion for the treatment of allogeneic acute rejection.Objective To investigate the effects of a combination of intravenous donor spleen cell infusion and reduced immunosuppressant administration on the allogeneic skin graft acute rejection in a mouse model. 2)To explore the possible mechanisms involved in the regulation of donor splenocyte infusion on recipient acute immunological rejection.Methods1. Optimization on the protocol of immunosuppressant-reduction induced acute allograft rejection in mice model. Allogeneic skin transplantation was performed between donor Babl/c mice and recipient C57BL/6 mice. 1)To determine the tolerable dosage of Cs A in C57BL/6 mice. Eighteen recipient C57BL/6 mice were randomly divided into three groups(n=6). Group?:intraperitoneal injection of Cs A, 50 mg/kg/day, until skin allografts were rejected; Group?: intraperitoneal injection of Cs A, 30 mg/kg/day, until the skin allografts were rejected; Group ?: intraperitoneal injection of Cs A, 20mg/kg/day, until the skin allografts were rejected. The postoperative changes of weight, and survival time of skin allografts(rejection was defined when 80% area of skin graft became necrostic). 2)Determine the optimal dosage-reduction of Cs A for inducing acute skin allograft rejection. Twenty-four recipient C57BL/6 mice were randomly divided into three groupst(n=8) and received Babl/c mouse skin grafts: Group A1(intraperitoneal injection of Cs A 30 mg/kg/day, until the the skin allografts were rejected; B1 group(recipients were given Cs A 30 mg/kg/day intraperitoneally for 10 days followed by administration of reduced dosage of CsA of 15 mg/kg/day), C1 group(received no Cs A). The mean skin allograft survival time of the three groups was campared and skin allograft were biopsied on postoperative days 3, 7, and 11 that underwent H&E staining and microscopic evaluation for immune rejection levels defined by Banff classification standard. The following statistical analysis methods were used: skin survival time was compared by the SNK- q test, the rate of two groups was compared by the student- t test, P?0.05 was considered to be statistically significant. 2. Experimental study on the immune regulatory effects of donor splenocyte infusion on immunosuppressant reduction induced acute rejection. Ninty-six C57BL/6 mice received Babl/c mice skin allograft transplantation and randomly divided into three groups: Group A2(positive control: intraperitoneal injection of Cs A) and Group B2(experimental Group: intravenous intraperitoneal injection of donor spleen cells + Cs A injection), Group C2(control Group: intraperitoneal injection of intravenous saline + Cs A), Group D2(negative control Group: recipients received donor skin grafts without Cs A administration). Cs A was intraperitoneally injected into recipient mice at a dose of 30 mg/kg/ after skin allografting, the dosage of Cs A was tapered to 15 mg/kg/day on postoperative day 10. Donor spleen cells or saline were injected on postoperative day 7. Postoperative the survival times of skin allograft between different groups were analyzed with SNK- q test, P?0.05 was considered to be statistically significant. On postoperative day 11,the skin allografts were biopsied and underwent H&E staining followed by microscopic evaluation for immune rejection levels. 3. Explore the possible mechanisms involved in the regulation of recipient acute immunological rejection by donor splenocyte infusion. The IL-2 m RNA level in recipient splenocytes of the above 4 Groups were assessed at postoperative days 1,4,7,8,10 and the IL10, TGF-? m RNA levels at day 11 of the recipient splenocytes were determined by quantitative PCR. The ratio of regulatory T cells in spleen cells on postoperative days 8, 10, and 15 were determined by flow cytometry.Results 1. The mice receiving Cs A50 mg/kg/day lost body weight starting from days 3 to 5 postoperatively and died at day 5. The mice receiving Cs A30 mg/kg/day demonstrated no obvious change of body weight and the average survival time of skin grafts was 18.4±0.5 day that was 3.0 day longer that the mice receiving Cs A 20 mg/kg/day with a statistically significant difference(P?0.05); The mean survival times of skin allografts of Group A1 and Group B1 were 18.5±0.52 day,11.12±0.4 day, respectively; The mean survival time of grafts in Group A1 showed 7.34 day longer than that of Group B1 with a statistically significant difference(P?0.05); According to the skin rejection pathology classification(Banff working classification), Group C1 showed level ?on postoperative day 7 while that in both Group A1 and Group B1 showed level 0. 2. The mean survival times of skin allografts of Group B2 and Group C2 were 14.3±1.02 day and 11.12±0.35 day, respectively, with 3.18 day longer in Group B2 relative to Group C2 with a statistically significant difference(P?0.05)According to the skin rejection pathological grading, pathological evaluation on H&E stained slides showed rejection morphology of level 0 in graft tissues of Groups A2 and B2 whereas level ? rejection morphology was observed in graft tissues of Groups C2 and D2. 3. The spleen regulatory T cell ratio in the mice of Group B2 increased significantly from days 8 to 10 while the other Groups showed no obvious change postoperatively. On postoperative day 10, the spleen regulatory T cell ratio of Group B2 was higher than that of Group C2(P?0.05). The IL-2 m RNA level of Group B2 elevated significantly from days 7 to 8 while that in the other Groups indicated no obvious change. On postoperative day 11, the spleen IL10, TGF-? m RNA levels of Group B2 were higher than that of Group C2(P?0.05).Conclusion Intravenous infusion of allogeneic spleen cells in acute rejection period after allogeneic skin grafting increases regulatory T cell ratio and the levels of inhibitory cytokines of IL10, TGF-?, leading to significant prolongation of allogeneic skin graft survival. |
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