| Background:Type1diabetes mellitus (type1diabetes mellitus, T1DM) is metabolic disease which serious threaten humans health, correlate to hypoinsulinism of patients. Islet transplantation can achieve noninsulin dependent and avoid diabetic complications occur and enhance long-term quality of diabetics, so, islet transplantation considered perfection methods for cure T1DM.So far, diabetics can not dependent on external insulin in14years after islet transplantation, and the grafts still have function. However, grafts function and quantity is proceed decrease after pancreatic islet transplantation, most of the patients long-term curative effect is not desirable.80%transplantation patients have C-peptide secretion after5years of islet transplantation, but only10%patients don,t rely on external insulin after islet transplantation. That demonstrate islets graft survival but function is inadequacy.Microvessel start occur in graft islets after7days of pancreatic islet transplantation, in7days, because of no blood supply,a bulk of islet apoptosis and death, only30%islets survival. Because of angiogenesis shortage, transplanted islet size is shrink compare to normal pancreatic islet,low density, low blood flow and low oxygen pressure, graft islets self-replication founction may be major factors. At present, most of scholars regards islet revascularization insufficiency as major factor affect long-term function after islet transplantation, immunologic rejection is not major factor that lead to graft islet low long-term effect.experimental evidence indicate that even transplantation syngenetic islet, long-term effect is also unsatisfactory,but could maintain normal blood glucose level in short-term. The research of self islets transplantation also support islet angiogenesis shortage is close to bad long-term effective. In order to islet transplantation widely use in clinic and obtain long-term stable effect, research the methods and mechanism to promote graft islet revascularization is efficient strategy to overcome islet revascularization insufficiency. It may be provide new ideal to therapy type1diabetes.Tumor transplantation model is common used to research animal model for tumor. Tumor cell growth in part, also form new blood vessels nets to support the tumor further growth. More and more research shows that the tumor mesenchymal cell plays a very important role in tumor angiogenesis, The stroma fibroblasts receive multiple angiogenesis factors effect could be activation by tumor secretion. Rise the key molecules to promote angiogenesis, Vascular Endothelial Growth Factor-A (vascular endothelial growth factor-A,VEGF-A),and also secretion Strong angiogenic factors, such as connective tissue growth factor (connective tissue growth factor,CTGF),transforming growth factor-β1(transforming growth factor-β1,TGFβ1) et al.So that the growth tumor,s new blood vessel and blood vessel，s density is increase.Base on the above reason,our research group focused on the activation or not activated fibroblast (FB) joint intervention of islet transplantation. Internal islet microvessel density may change, preliminary discussion the mechanism of fibroblasts promote graft islet microvessel happen.Objective:Explore each experimental groups islet internal microvessel density change after pancreatic islets combine activated/not activated fibroblasts transplantation; Explore the islets microvessel density increase whether from donor or receptor fibroblasts plays a major role after pancreatic islets combine activated/not activated fibroblasts transplantation.Methods:This study is a new experimental model can be used in clinical pancreatic islet transplantation. The tumor cells culture supernate fluid activated fibroblasts and testing (abbreviation,Xiao Heng provides).Use C57BL/6mice, male (6-7week);Divide groups:group A:simple islet (H2d) transplantation; group B: islet (H2d) combine donor FB (H2d) transplantation; group C:islet (H2d) combine receptor FB (H2b) transplantation; group D:islet (H2d)combine donor activated FB (H2d) transplantation; group E:islet (H2d) combine receptor activated FB (H2b) transplantation; group F:syngenetic islet (H2b) transplantation.T1DM model could be induct by intraperitoneal injection streptozocin (STZ). Pancreatic islets combine activated/not activated fibroblasts renal capsule transplantation. Under the same conditions, use Collagenase P solution with pancreatic duct perfuse, discontinuous density gradient purification methods obtain mouse islet by Ficoll-400. Islet morphology and purity detection by Dithizon (DTZ), Islet survival rate detection by0.4%trypan blue. Islet secret function detection by mouse insulin ELISA kits. Mouse blood sugar change after transplantation detection by fast blood sugar test strip. Ensure transplant islets have high quality. Different Time Periods (5day,14day and30day) intislet microvessel density is detection by Immune fluorescence and confocal.and the histological changes of transplant pancreatic islets in different groups were observed by HE stain in different time (each groups transplant pancreatic islet specimens were obtain at the islets no function, group A, B, C, D, E and group F obtain the specimens time was14,14,14,30,100and100day). Allogeneic transplant mice (group A, B,C, D and E) adopt Tacrolimus and Sirolimus antirejection therapy.Results:Tumor cells culture supernate fluid have certain promote effect to fibroblasts.Tumor cells culture supernate fluid composed condition medium can promote α-SMA and VEGF-A expression in fibroblasts (abbreviation). Successful isolation and purification mouse pancreatic islet. Each acceptor can obtain900±30Islet equivalent (IEQ),each BALB/c donor mouse average obtain350±25IEQ, purity is85%±5%;each C57BL/6donor mouse average obtain324±18IEQ, purity is81%±4%;pancreatic islet average survival rate is97%±2%;also pancreatic islet secretory function is well (SI>2). Hyperglycemia turn to normal after pancreatic islet transplantation. There was no significant difference in the blood glucose between each groups mice before transplantation (P>0.05),all mouse blood glucose under11.0mmol/L in3day after transplantation, there was no significant difference in each group (P>0.05).There was no significant difference in the decreased value of blood glucose between three groups (A,B,C)(P>0.05).There was no significant difference in the decreased value of blood glucose between group E and group F(P>0.05),but the decreased value of blood glucose in group E is lower than group F. There was significant difference in the decreased value of blood glucose in group D compare group A, B, C, also compare group E and group F (p<0.05).The mice average survival time in group A,B,C,D,E and group F in proper order was13±1day,15±2day,15±2day,29±2day,>100day,>100day. There was no significant difference in the microvessel density between each group at5day after transplantation (P>0.05). There was significant difference in the microvessel density between each group at30day after transplantation (P<0.05).HE stain:there was serious inflammatory cell infiltrate in transplant islet of group A, group B and group C, part islet shape was broken down, and little inflammatory cell infiltrate in transplant islet of group D, group E, regular shape, round or oval, cytoplasm and karyon clear identify, and multinuclear, there was regular shape, cytoplasm slightly stained and multinuclear, islet survival well.Conclusion:In the current experimental and clinical stage, this research provide a new thinking to revascularization in pancreatic Islets transplantation;Relative to the simple pancreatic islet transplant and islet combine nonactivated FB, activated FB have well function in revascularization, especially of receptor FB, whether immune system of receptor reject donor source FB we were unknown;Activated FB of receptor combine pancreatic islet transplant can cure diabetes, also can long-term survival;Under the transplantation of pancreatic islet combine activated FB of receptor, whether the pancreatic islet quantity may reduce, and the least pancreatic islet quantity have require we were unknown, require further study. |
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