| BackgroundDiabetes mellitus (DM) . one metabolic disorder disease including T1DM or part of T2DM. is caused by autoimmune and environmental triggers, with either absolute or relative insulin deficiency due to the selective destruction of insulin-producing pancreatic beta cells. DM is a leading cause of morbidity and mortality in all over the world. Its treatment is one of the great challenges for the modern medicine during the 21st century. However, with the rapid advances in modern biomedical technology, exogenous insulin treatment can precisely control the blood glucose levels, but some chronic complications related to DM and even the risk of hypoglycemic events affect the quality of life of patients. Pancreatic islet transplantation is a promising method to restore normoglycemia in recipients undergoing treatment of DM. However, the infusion of sufficiently large quantities of islets is one of the major obstacles for the widespread application of islet transplantation. ObjectivesTo observe how to improve the techniques for pancreatic islet extraction , so as to acquire enough amount and also high quality of transplantable cells for clinical islet transplantation. Using different techniques, rat islet samples were isolated, purified, then were allotransplanted under the kidney capsule of diabetic SD rats induced with streptozotocin(STZ), respectively, without any immunosuppressive agents. Meantime, an comparison of the time for reverse hyperglycemia in islet recipients was carried out. Our data could supply some experimental parameters for the consideration of clinically application of islet transplantation. Material and MethodsUnder the same experimental conditions, pancreatic islets from SD rats were isolated, digested with the injection of Hank's (including 1mg/ml collagenase V and 2.5% glycerin) into the common bile duct in group A and B (both, n=6), or this injection was omitted in group C (n=6). Then, the pancreatic tissues were directly cut into small pieces, digested, purified with Ficoll discontinuous density gradient centrifugation in group A and C, or by filtration with a hand-held prewet 100 μm stainless steel sieve in group B. All islet cell masses from over 50μm in diameter was counted . Islet samples were stained with dithizone(DTZ) for estimation purity of islets, and Cell viability was assessed by trypan blue exclusion. Islet grafts were processed with routine paraffin section for further detection under light microscopy.By using different techniques in group A(n=6) and B(n=6), rat islet samples were isolated , purified , then were allotransplanted under the kidney capsule in group D (n=6)and E(n=6) of diabetic SD rats induced with streptozotocin(STZ) , respectively, without any immunosuppressive agents. Meantime, an comparison of the time for reverse hyperglycemia in islet recipients was carried out. ResultsThe result of islets before were purified, Group A (n=6), the average islet yield was 562±59islets/pancreas, purity was 43.7±4.0%, viability was (97.5±1.5)%; Group B (n=6), the average islet yield was 600±62 islets/pancreas, purity was 43.9±3.2%, viability was 97.0±1.2%; Group C (n=6), the average islet yield was 346±41 islets/pancreas, purity was 43.1±2.6%, viability was 96.3±1.3%. After purification, Group A, the average islet yield was 352±33 islets/pancreas, purity was 83.7±2.1%, viability was 91.0±1.3%; Group B, the average islet yield was 494±56 islets/pancreas, purity was 53.0±3.7%, viability was 94.7±1.9%; Group C, the average islet yield was 236±24 islets/pancreas, purity was 80.6±2.4%, viability was 82.9±2.2%. Our data revealed that, before purification, there were no significant differences in their yield and quality of islets in group A between group B with the same method for isolation of rat islets, the yield of islets was higher in group A or B than in group C with the different method for isolation of rat islets (P<0.01) , respectively; and there were no significant differences in the purity and viability. With the same purification method in group A and C, and another method in group B, the yield and viability of islets was higher in group B than in group A (P<0.01) ; and the lowest in group C. However ,the purity was higher in group A than in group B or C (P<0.01 ,P<0.05) , respectively; group C was significantly higher group B(P<0.01).The pancreatic islets isolated from Group A & B were transplanted into the diabetic SD rats in group D(n=6) & E(n=6) under the rat renal capsule in our experiment, respectively. Recipients' blood glucose was monitored, the initial time of hyperglycemia reversed to normalize glucose 48-72 hour (group D, the average time was 64±9.8 hour ) and 36-48 hour (group D, the average time was 42±6.6 hour ) after transplantation ( P<0.01 ) , respectively. Without any immunosuppressive agents, the islet grafts was excluded 10.7±2.0 days in group D and 6.8±1.9 days in group E posttransplantation follow-up. The data revealed that the time of hyperglycemia reversed in the diabetic rat was longer in group D than in group E (P<0.01) . Than all of the islet grafts was removed from the renal subcapsular space, their structure was observed with lightmicroscopy including the modality of islets and any islet capillary formation, there were no differences in structure between islet grafts and endogenesis rat islet of control group, and that islets can function normally after engraftment into the kidney subcapsular space. Conclusion1. The reasonable method of isolation and purification of the islets is that injecting Hank's solution (including lmg/ml collagenase V and 2.5% glycerin) into the common bile duct with 22 or 24 size trocar, then isolating the rat pancreas when it expanded adequately.2. The yield and viability of purified islets was significantly higher by filtration with a hand-held prewet 100 μm stainless steel sieve than by Ficoll discontinuous density gradient centrifiigationor, and the islet graftments more early survival and hyperglycemia reversed than the latter ones posttransplantation, suggesting this method can be used to apply for clinically pancreas islet transplantation.
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