Pre-Clinical Research Of Islet Transplantation For Diabetes Therapy

Pre-Clinical Research of Islet Transplantation For Diabetes therapyObjective Diabetes mellitus is one of the diseases to harm seriously for human health. Islet transplantation has been demonstrated to be an effective therapy for insulin-dependent diabetes. This research is to establish the protocols for human islet isolation and purification and to evaluate the functions and safety of human islet products in vitro and in vivo as well as eventually to provide experimental basis for clinical application of islet transplantation.Methods Six human pancreases were obtained from the 181st hospital of PLA. Human pancreases were digested using automatic method established by Ricordi and purificated by the Ficoll density gradient centrifugation in COBE 2991 blood cell processor. The islet counts and purities were calculated by a computer picture analysis program in a microscope (Nikon, SMZ1500) after DTZ staining. The mRNA expressions of insulin, glucagon and several islet relegated autoantigens were detected by RT-PCR. The glucose-induced insulin secretion was detected in vitro by chemiluminescence method. Insulin secretory granules of the isolated islets were observed by electron microscope. The cellular composition and distribution of the isolated islets were checked by a fluorescent microscope. The vitality of the isolated islets was analyzed by a computer picture analysis program after AO/PI staining. The insulin secretion function of the isolated islets was further evaluated in vivo by islet transplantation in diabetic nude mice. The medium of the islet products were cultured for detecting aerobic, anaerobic bacteria and fungus. The endotoxin content in the medium of the islet products was tested by TAL gel formation method. The procoagulant activity in medium of the islet products was determined by clotting time.Results The cold ischemia time of the pancreas donors was 10 to 12 hours. The mean weight of these pancreas was 65.61g±19.31 g(41.30g-94.87g). The mean count of the islets was 229,000±31,000 IEQs (4970±1620 IEQs/g tissue) and the mean purity of the islets was 59.0%±8.9% (49%-70%) after purification. The vitality of the islets was 89%±3% (85%-94%) by AO/PI staining assay. By RT-PCR, the mRNA expressions of insulin, glucagons, IA2, IAR, ICAp69 and GAD65 were positive in the islets. The static insulin releasing test indicated that the mean insulin stimulation index was 8.1±4.0 (3.8-10.2) . By transmission electron microscope, a great quantity of insulin secretory granules was found in the cytoplasma of islet cells. The result of immunofluorescence staining showed that C-peptide positive cells were mainly distributed in the central of the islets, glucagons positive cells were mainly located the perimeter of the islets and polypeptide positive cells were distributed scatteringly in the islets. The vitalities of islets before freezing and after thawing were 90% and 85% respectively by AO/PI staining assay. The insulin stimulating index of the islets after thawing was 2.5. In order to evaluate function of the human islets, isolated human islets were transplanted under the capsule of left kidney in STZ induced diabetic nude mice. The result indicated that the glycemia of the diabetic nude mice decreased to normal levels on the third day after islet transplantation and maintained for a long-term, while the glycemia of the mice increased to diabetic state after the left kidneys with grafted islets were removed. The culture medium of islet products was collected to test bacteria and endotoxin. No aerobic or anaerobic bacteria and fungus was detected in the culture medium of islet products. The levels of endotoxin in the medium of islet products were below 0.5 EU/ml and procoagulant activities in that were below 300U/ml.Conclusion We have established a practical protocol of human islets transplantation. According to this protocol, we have obtained adequate amount, high purity, functional and safe human pancreatic islets products for clinical islet transplantation. Our research works establish abundant experimental foundation for clinical islet transplantation in the future. The experimental study of pancreatic stem cells differentiating into insulin producing cellsDiabetes mellitus is a chronic metabolic disorder disease characterized as hyperglycemia that results in several serious complications such as diabetic nephropathy, diabetic retinopathy, et al. Morbility of diabetes is increasing greatly worldwide these years. The diabetic complications threaten human health greatly and causing death and disability. Insulin injection therapy can not control metabolic disorder perfectly and can not reverse the diabetic complications. Pancreatic islet transplantation is an effective therapy to cure insulin-dependent diabetes mellitus (IDDM) . Howerver, the deficiency of pancreas donors limits the clinical application widespreadly. Stem cells own the potency of proliferation and differentiation into multi-kinds of tissue cells. Induction stem cells to differentiate into endocrine cells of pancreatic islet will possiblely solve the deficiency of pancreas donors.Objective In the research, the pancreatic stem cells were isolated and purified from rat pancreas. Then the stem cells were induced to differentiate into pancreatic endocrinal cells and to form islet-like structures. To evaluate the potential application of stem cell for diabetes therapy, the function of these stem cell induced islets was further evaluated in vitro and in vivo.Methods Firstly, rat islets and pancreatic duct cells were isolated from rat pancreas by collagenase digestion and density gradient centrifugation. Then, pancreatic stem cells were purified from pancreatic duct cells using nestin conjugated magnetic beads. The pancreatic stem cells were induced proliferation, differentiation and formation of islet-like structures by inductors such as insulin-transferrin-selenium, nicotinamide, basic fibroblast growth factor and following by suspension culture in additive extracellular matrix including fibronectin, laminin and collagen type IV. The function of these stem cell induced islets was further evaluated in vitro and in vivo.Results Pancreatic islets and pancreatic duct cells were isolated from rat pancreas by collagenase digestion and density gradient centrifugation. Then, the pancreatic stem cells were purified from cultured pancreatic duct cells using nestin conjugated magnetic beads. After proliferation, the stem cells were induced to differentiate into insulin producing cells by inductors such as insulin-transferrin-selenium, nicotinamide and basic fibroblast growth factor and to form islet-like structure following by suspension culture in additive extracellular matrix including fibronectin, laminin and collagen type IV. The expression of insulin mRNA in the stem cells differentiated islets was significantly up-regulated by reverse transcription polymerase chain reaction (RT-PCR). Immunofluoresence staining indicated that lots of insulin positive cells and a few of glucagon positive cells were existed in the islet-like structures. The glucose-induced insulin releasing in stem cell differentiated islets was 39.4% of that in native rat pancreatic islets. In addition, the glycemia in diabetic nude mice was normalized after transplantation with the stem cell differentiated islets.Conclusion: The nestin positive stem cells derived from rat pancreas could be induced differentiating into insulin producing cells, which exhibited the potential application for diabetes therapy.