The Experimental Study On The Isolation And Purification Of The Islet Cells From Human Pancreata

Background Islet transplantation has the potential to cure diabetes mellitus. Nevertheless despite successful reversal of diabetes in many small animal models, the clinical situation has been far more challenging. New hope for the treatment of type 1 diabetes has recently emerged from the encouraging results of islet cell transplantation in humans known as "The Edmonton Protocol" during the last few years. However the success of clinical trials is still limited by the inability to transplant enough viable human islets to compensate for the insulin-deficient state, the number of islets that engraft following transplantation, the rejection process, and the recurrence of autoimmunity. The advances in immunosuppressive regimens, organ procurement techniques, isolation techniques, and availability of defined collagenase blends have contributed to the continuing promise of making islet cell transplantation the treatment of choice for type 1 diabetes mellitus. The aim of this study is to explore the method of isolation and purification of the islet cells from human pancreata and examine the role of some immunosuppressive agents on islet cells in vitro following isolation and purification.Objective 1. To establish an effective method for isolation and purification of the islet cells from human pancreata by using a two-step digestion and tube purification method. 2. To investigate the ability of Pefabloc and PNGS improving the quality and yield of islets from human pancreata. 3. To evaluate the role of three kinds of immunosuppressive agents on islet cells in vitro by MTT method following isolation and purification.Method 1. Isolation and purification of islet cells: With our own method, pancreatic tissue was enzymatically digested by two-step digestion method and purified by discontinuous gradient centrifugation on four layers of HC-A-Ficoll solutions by tube method. 2. After isolation, evaluating the islet yield and purity with the light microscope by staining with DTZ, assessing islet function by insulin release assays in vitro. 3. Immunohistochemistry and observations with transmission electron microscope were performed to identify the insulin and glucagon expression in islet cells. 4. Counting the islet number in Pefabloc and PNGS groups by using DTZ staining with the light microscope after isolation and assessing islet function by insulin release assays in vitro. 5. Evaluating the activity of islets by MTT assay after incubation with three lands of immunosuppressive drugs at different concentrations following isolation and purification.Results 1. After the purification step by discontinuous gradient centrifugation, about 81444+34631 islets per pancreas and 1136±474 islets per gram of pancreas were obtained from human pancreata, with an average purity of 74.84%. 2. The purified islets were respond to high concentration glucose stimulation(300mg/dl) with 2.15 times increase of insulin secretion over basal levels(60mg/dl), the difference was significant(p<0.01) , and the islet cells contain insulin and glucagon granules. 3. The yield of purified-5-islets was significantly higher in Pefabloc groups(1559±319 islets/gram of pancreas) compared with that in Control groups(750±267 islets/gram of pancreas) and the difference of SI between Pefabloc groups(1.93±0.21) and Control groups(1.86±0.19) was no significant; the difference of the yield of purified islets was no significant between PNGS groups(871±204 islets/gram of pancreas) and Control groups(750±267 islets/gram of pancreas) and SI increased significantly in PNGS groups(2.74±0.54) than in Control groups(1.86±0.19). 4. The activity of the islet cells was not reduced after exposure to rapamycin with lower concentration and significantly inhibited with higher concentration(> 1ng/mL). No reduction of the islet's activity was observed after exposure to daclizumab and FTY720 with either lower or higher concentration.Conclusion 1. Establish a method for isolation and purification of the islet cells from human pancreata. 2. Pefabloc can imp...