| Background As a potential method to cure diabetes mellitus, islet transplantation has got much attention long-termly, especially after the success of Edmonton Protocol which exploited an extensive application perspective to cope with type 1 diabetes. However, the protocol still require immunosuppressive agents to suppress transplantation rejection, which must bring much side effect and underlying risk to patients, for instance, increasing the chance of infection or tumor. Some immunosuppressive regimens are harmful to islet cells which could cause diabetes mellitus after transplantation. The methods of inducing islet transplantation tolerance would prolong the survival of islet grafts after transplantation because the immune tolerance can make patients avoid the toxic effects of continuous immunosuppressive treatment. Some recent researches demonstrated that the apoptotic cells can actively regulate immunity function of organisms and induce immune tolerance by means of regulating cell immunity and humoral immunity. The apoptotic cells can induce immune tolerance because they can inhibit the maturation of immature Dendric Cells which couldn't provide enough co-stimulation signals to activate T cells, and because phagocytes produce immunosuppressive factors after phagocytosing apoptotic cells. Therefore, to investigate the method of prolonging islet survival, we would find an effective way to induce islet transplantation tolerance through the apoptotic cells.Objective To investigate whether the transfusion of donor apoptotic cells could suppress islet grafts rejection and prolong their survival time. To provide experimental evidences for establishing protocols inducing islet transplantationtolerance.Methods1. To investigate the influence of hydrogen peroxide on the human spleen cell in vitro: the human spleen cells obtained by the method of grinding were divided into four groups and treated with saline solution or various concentrations of H2O2 respectively. Mitochondrial function was assessed with the 3-(4,5-dimethiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT) and the early apoptotic cells were detected by flow-cytometry with Annexin V-FITC/PL;2.Isolation and purification of human islets: to digest pancreases with semiautomat pancreatic segregation apparatus and to purify islet cells by COBE-2991 cell separation machine via discontinuous gradients centrifugation;3.To establish animal model of diabetes mellitus: KM mouse were rendered diabetic via single intraperitoneal injection of streptozotocin at 120mg/Kg weight(continuous twice blood glucose>20mmol/L);4. To study the effect of transfusion of donor apoptotic cells on mixed lymphocytes culture: Diabetic KM mouse were randomly rendered infusion of Hanks, normal donor cells, apoptotic donor cells and necrotic donor cells, then, 7 days after the infusion, all recipients' spleen were resected and the isolated lymphocytes were co-cultured with donor lymphocytes. The results of mixed lymphocytes culture were observed via MTT method;5. To study the effect of transfusion of donor apoptotic cells on the survival of islet grafts: Diabetic KM mouse were randomly rendered infusion of Hanks, normal donor cells, apoptotic donor cells and necrotic donor cells, then, 7 days after the infusion, all mouse received islets (lOOOIEQ/a STZ KM ) under their renal capsule respectively and the survival time of islet grafts were compared;6. Grafts from recipients were examined for the expression of insulin by imrnunohistochemical staining at the time of euglycemic or returning to a hyperglycemic state in different groups.Results1. The Mitochondrial function and the percentage of apoptosis of the human spleen cells showed a dose-effect relationship with H2O2.The former descended with the increase concentration of H2O2 while the latter demonstrated a contrary tendency.Moreover, they had a time-effect relationship with the treatment. There were significant different rates of the apoptotic cells within groups (P< 0.05).The cells treated with H2O2 in the concentration of lOOumol/L reached the highest percentage of apoptosis(63.95 ±4.11)% at the 6th hour after treatment.;2.After the purification step by discontinuous gradient centrifugation, about 9.63±7.09 ten thousands IEQ per human pancreas were acquired, with an average purity of (34.4±14.0)%,and the purified islets were respond to high concentration glucose stimulation with 2.31 times increase of insulin secretion over basal level;3. Pharmacological diabetes on KM mouse can be induced successfully by single intraperitoneal STZ injection (120mg/Kg weight) with an average model-performed rate of 90%;4. After the one-way mixed lymphocyte culture, the proliferative response of lymphocytes derived from diabetic KM mouse who received donor apoptotic cells to donor lymphocytes was apparently decreased, compared with who received Hanks, normal donor cells and necrotic donor cells, and the stimulation indexes were 1.46±0.16 , vsl.90±0.38, 1.82±0.22, 2.10±0.22 (P< 0.05);5. The survival time of islet grafts of diabetic KM mouse who were pretreated with donor apoptotic cells were dramatically prolonged, compared with who were pretreated with Hanks, normal donor cells and necrotic donor cells, 19.6±7.0days, vs5.6±0.9> 6.0±1.6> 5.5±1.0days(P< 0.05);6. Graft tissue from groups who were pretreated with Hanks, 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 KM mouse 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 diabetic states, showed no staining for islets.Conclusion 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.. |
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