| Background and purpose The studies of animal and clinic researches oflimb allotransplantation have got a great development. In comparison with other organ transplantations, limb allotransplantation has some characteristics. Being composite tissue, limb's immunogenicity and rejection are stronger than other organs. Because the limb allotransplantation has relation with bone marrow allotransplantation, so the immunosuppression in limb allotransplantation is harder to achieve than other organ allotransplantation. As a result, route immunosuppressant with common plan and dose is difficult to get a good effect. So, the recipients have to take medicine for long time, have to pay for more money and face such side-effects as liver and kidney damages, chance of infection and tumor. The researchers have come to a common understanding the better effect could to be achieved by several methods according to every step in rejection. As a central immune organ, thymus operates on T cell's growth, differentiation and selection. Thus, after being injected with antigen, the recipient's immune tolerance could be achieved. Thymocyte after positive selection identifies the complex consisting of major histocompatibility complex(MHC)and self antigen and regard the complex as his own antigen. As a result, the thymocyte identifying the complex can be deleted. At last, the immune tolerance is built. Recently, some researchers reported that thymus is a immunologically privileged organ and there is not rejection after allotransplantation init. This phenomenon is related to apoptosis. Because the cell in the immunologically privileged site gives high expression of FasL, when the transplant cell enters, the peculiar T cell which can identifies the transplant cell is activated, but the peculiar T cell gives high expression of Fas antigen and apoptosis through Fas/FasL pathway. As a result, the recipients have the ability to accept the donors without rejection. The splenocytes have plenty of dendritic cells which belong to lymphatic dendritic cell (LDC) and have a great deal of MHC on their surface. While the donor-strained splenocytes were injected into the thymus, they can induce the T cell to produce negative selection. At last, the T cell stops growing and the immune tolerance is built. In the present study, we injected donor-strained splenocytes into recipient's thymus and expected to induce immune tolerance. After the limb transplantation, we observe the circulation of the transplantate limb, the rejection time and the survival time of limb grafts, detect the pathological changes of limb grafts and measure the levels of IL-2 and IL-10 of the recipients. At last, we hope to provide a new way to prolong the survival time of limb grafts.Materials and Methods A total of 16 male SD rats were randomlydivided into two groups: the control group(n=8) and the experimental group(n=8), and used as recipients. A total of 8 male Wistar rats were randomly divided into two groups and used as donors. The one half of donors' spleen in two groups was removed. After the operation, the spleens were chipped into cell suspension which has 2X 108 cells in one milliliter, at the same time, 95 percents of this splenocytes were live. The recipients in experimental groups were injected with donor-strained splenocytes with total number of 4X107, and the recipients in control group were injected with placebo. All recipients were injected with cyclophosphamide into abdominal cavity after the injection(150mg/Kg). Two weeks later, Wistar to SD rats limb allotransplantation was performed. Each donor provided two limbs to two recipients. The rejection time and the survival time of limb were investigated. The histologic degrees of skin and muscle of limb allograft at 3, 5, 7, lOd after allotransplantation were detected according to Buttemeyer and Lee's standards. Thelevels of IL-2 and IL-10 of the recipients before and after induction and 3, 5, 7, lOd after allotransplantation were also measured by enzyme linked immunosorbent assay(ELISA). SPSS 10.0 software was used to analyze the data, P<0.05 was considered the level of significance.Results 1. The rejection of limb allograft in the experimental group andcontrol group took place after limb allotransplantation. 2. After limballotransplantation, the quantity of food and movement of all recipients in the experimental group and control group was smaller than that of normal rat. The signs of graft-versus-host disease(GVHD) such as anorexia, diarrhoea and apathetic ileuswere not found. 3. The first signs of rejection of limb allograft in the experimentalgroup and control group was dropsy and erythema on skin which often appeared on the lateral shin of the thigh. The first symptom of putrescence of limb allograft was nigrescence, scab and effusion emerged on skin. In the experimental groups, the mean rejection time of limb allograft was 7.63 ± 1.85d, the mean survival time of limb allograft was 15.38 ±2.97d. On the contrast, in the control group, the mean rejection time of limb allograft was 5.00 +1.3Id, the mean survival time of limb allograft was 9.38 + 1.92d. The mean rejection time and survival time of limb in the experimentalgroup was longer than that in the control group (/?<0.05) . 4. The histologicsymptoms of skin in the experimental groups and the control group included variable basal cell vacuolation and necrotic keratinocyte and lymphocytic infiltrateing in the upper dermis often extending into the dermis, increasing severity of basal cell vacuolation leading to bulla formation, necrosis of the epidermis with variable mixed cell infiltrates. At the same time, the histologic symptoms of muscle in the experimental group and the control group included patchy focal mild mononuclear interstitial infiltrating without muscle necrosis, progressive increase in the numbers of mononuclear cells occurring in between myositis and perivascular region, an aggressive diffusing lymphocytic infiltrating, muscle necrosis, endothelitis, and interstitial edema. The histologic degrees of skin and muscle of limb allograft at 3, 5,7, lOd after allotransplantation in the experimental group was lower than that in the control group (p <0.05) 5. There was no significant difference in the content of the IL-2 and IL-10 of the recipients before the induction in the experimental group and control group (/?>0.05 ) . The content of the IL-2 at 3, 5, 7, lOd after allotransplantation in the experimental group was lower than that in the control group (p<0.05) , at the same time points, the content of the IL-2 in the control group increased higher than that in the experimental group. The content of the IL-10 at 3, 5, 7, lOd after allotransplantation in the experimental group was higher than that in the control group (p<0.05) , at the same time, the content of the IL-10 in the experimental group increased higher than that in the control group.Conclusion 1. Wistar to SD rat limb allotransplantation could be regard as aperfect model of limb allotransplantation. This model have the advantages of the rejection severity and invariableness, easy to observe the limb allograft, having enough peripheral blood to take several measurements, easy to take histologicexamination of limb allograft. 2. Intrathymic injection of donor-strained splenocytes in the lamb transplantation can obviously prolong the survival time of limb allograft. Several measurements could show that the immune tolerance could set up. 3. The immune tolerance induced by intrathymic injection of donor-strained splenocyte is imperfect and temporary.
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