| BackgroundIslets transplantation to re-establish the secretion system of insulin has been considered as a most promising treatment to cure diabetes mellitus. Especially the success of Edmonton Protocol in 2000, has demonstrated the great progress which has been made in the territory of islets transplantation, and accordingly exploited an extensive application perspective of islets transplantation to cope with type 1 diabetes. However, transplantation rejection and the potential risk coming with the application of immunosuppressive agents are still the obstacles unable to overcome completely. Now it is generally thought that the key to solve the rejection of allografts is to induce the recipient's immune tolerance to the grafts from the donor. Though there are so many means to induce the tolerance, none of them can achieve the complete permanent specific tolerance. Apoptosis is a kind of physiological or pathological phenomenon generally existing in the biosystem, and plays an important role in maintaining the balance and stabilization of the host's immune state. Apoptotic cells can secrete lipidic haemostatic factors , and present "eat-me" signals on the surface to attracted the phagocyte to remove themselves; at the same time, antigent-presenting cells (monocytes and macrophages, etc ) can secrete many inhibitive immune factors such as TGF-β,PGE2,IL-10 etc, after the phagocytosis. All of these creat a microenvironment for antigen recognition and induce the related lymphocytes' tolerance to apoptotic antigen rather than the inflammatory response. On these grounds, Sun et al proposed to make use of the transfusion of apoptotic cells to induce the donor's specific tolerance. Our workgroup has demonstrated that apoptotic cells can make an inhibitive effect on the proliferation of T lymphocytes in vitro, and researchers have reported that transfusion of apoptotic cells can notably prolong the survival time of rat allografts after the heart transplantation or liver transplantation. So we plan to induce the allogenic islets transplantation tolerance in diabetic SD rat by pre-transfusion of apoptotic cells to investigate the method of prolonging the survival time of the islets grafts.ObjectiveTo investigate the methods and mechanism of islets transplantation tolerance induced by transfusion of the apoptotic cells. To provide experimental evidences for establishing new protocols which can induce islets transplantation tolerance.Methods1. To investigate the effect of X-irradiation from electron linear accelerator on the rats spleen cell in vitro: the Wistar rat spleen cells obtained by the method of grinding were divided into four groups: control (A) and B, C, D group were irradiated by the absorbed dose of 1.5Gy, 2.0Gy, 3.0Gy respectively. And then incubated in 37℃, 5%CO2. The early apoptotic cells at 4h, 8h, and 12h were measured by flow cytometry (FCM) with Annexin V-FITC/PI.2. Isolation and purification of rat islets: The pancreatic islets were digested and isolated by perfusing collagenase P via pancreatic duct and purified by Ficoll-400 discontinuous density gradients after l-2d incubation.3. To establish animal model of diabetes mellitus: SD rats were rendered diabetic via single intraperitoneal injection of streptozotocin at 56mg/kg ? weight (continuous twice plasma glucose >16.7mmol/L).4. To study the effect of transfusion of donor apoptotic spleen cells on the survival of islet grafts: Wistar and SD rats were taken respectively as donor and recipient. Diabetic SD rats were randomly divided into 4 groups, and recived infusion of Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells (respectively as A, B, C, D group) via dorsal vein of penis, then, 7 days later, all rats received islets (1000IEQ per STZ SD rat) under their left renal capsule respectively and the survival time of islet grafts were compared. The insulin lever in serum were measured by RIA at 0d, 7d, 14d, after transplantation and at the time of rejection. Grafts from recipients were examined for the expression of insulin by immunohistochemical staining at the time of euglycemic or returning to a hyperglycemic state in C group. Grafts of other groups were examined for the expression of insulin by immunohistochemical staining only after the rejection.5. To observe the effect of transfusion of donor apoptotic cells on the Proliferative response of the lymphocytes from the recipient rats: Diabetic SD rats were randomly rendered infusion of Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells, then, at 0d, 7d, 14d after transplantation and at the time of rejection, all recipients' spleen were resected and the isolated lymphocytes were stimulated with ConA. Then the extent of the Proliferative response of the recipient's lymphocytes were observed by the method of cell staining with CFSE.Results1. The Wistar rat splenocytes were induced by X-irradiated with accelerator linear: the ratio of apoptotic cells in 2.0Gy group has a difference in the different time group (F=5.930,P=0.026). And has a difference compared with the control and 1.5Gy group (P<0.050) . The 2.0 Gy group has a highest Apoptosis rate at the time of 8 h after the Irradiation.2. After the purification by discontinuous gradient centrifugation, about( 1063.49±84.74) IEQ per rat pancreas were acquired, with an average purity of (70.51±6.20)%, and the purified islets were responded to high concentration glucose stimulation with 2.72 times increase of insulin secretion compared with low concentration glucose stimulation. 3. Pharmacological diabetes on SD rats were induced successfully by single intraperitoneal STZ injection (56mg/kg·weight) with an average model-performed ratio of 94%.4. The survival time of islet grafts of diabetic SD rats who were pretreated with donor apoptotic cells were dramatically prolonged, compared with who are pretreated with Hank's, normal donor cells and necrotic donor cells (P<0.050), with an average survival time of 32.00 days and the longest survival time of 42 days, the normal donor cells group also has a prolonged survival time compared with the A ,D group. The A, D groups had no prolonged effect.5. The insulin level in serum of the diabetic SD rats in C group had a notable increase at 7d, 14d, compared with the time of pre-transplantation (P<0.050), and the insulin level in C group at 14d after transplantation was higher than the B group (T=3.439 ,P=0.009) . The A, D groups have no significant changes.6. Grafts from groups who are pretreated with Hank's, normal donor cells and necrotic donor cells, harvested at the time of return to the diabetic state, revealed the complete absence of insulin-positive cells. Compared with control grafts, the grafts from STZ SD rats being pretreated with donor apoptotic cells, harvested from euglycemic animals, showed intact islets immunohistochemically stained for insulin. In the contrast, the grafts harvested from the same group at the time return to hyperglycemia, showed no staining for islets.7. After the stimulation by ConA , the Proliferative index of lymphocytes before transplantation and at 7d after transplantation in C group was lowest compared with other groups (F=29.943, P=0.000) . and the C group had a lowest proliferative index before transplantation(P<0.050). the suppressing effect existed until at 14 days after transplantation and disappeares after the rejection.Conclusion1. Irradiation by X rays from electron linear accelerator is a convenient, safe and effective way to induce apoptosis of Rat spleen cells. 2. A large quantity of islets which still have good functions can be acquired by isolation by intraductal collagenase P and purification by Ficoll-400 discontinuous density gradients after culturing 1 -2d .3. The transfusion of donor apoptotic cells can prolong the survival time of islet grafts and suppress transplantation rejection; therefore, the utility of donor apoptotic cells will be a feasible pathway to induce islets transplantation tolerance.4. We have demonstrated that the transfusion of donor apoptotic cells can have an inhibitive effect on the proliferation response of the recipient's lymphocytes to the stimulation of Con A. |
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