Transplantation Of Allogeneic Pancreatic Islets And Followed By Pancreatic Stem Cell Derived Islet-like Structures To Treat Diabetes

Objective:To Study the transdifferentiation of allogeneic pancreatic islets and followed by pancreatic stem cell derived Islet-like structures to treat diabetes. The allogeneic pancreatic islets and followed by pancreatic stem cells derived Islet-like structures were grafted under the kidney capsule.Blood glucose level and health state were mornited.to observe the efficacy of Transplantation of allogeneic pancreatic islets and followed by pancreatic stem cell derived Islet-like structures to reverse the diabete. Which may provide the experimental evidence for application of clinical treatment of diabetes.Methods:Rat pancreas was digested with collegenase followed by non-continuous density gradient to separate islets from exocrine tissue. Duct epithelial cells which are potential stem cells were cultivated in CMRL1066 and then in serum-free DMEM/F12 medium with the addition of nutrition factors for up to 27 days. Samples were taken at different time points(1,3,5,7,14,21,27d) For light and electron microscopy and for immunocytochemistry with antibodies against PDX-1 and CK-19. Diabetic rats were induced by injection of STZ(60mg/kg) into abdominal cavity. Rats with the blood glucose higher than 16.7 mmol/L for two consective days were taken as the models of diabetes.The models of diabetes were selected and randomizely divided into 3 groups: pancreatic islets transplantation group (n=13, groupA):rats in this group were implanted with 200±50 islets under the kidney capsul; Pancreatic islets and followed by pancreatic stem cells derived Islet-like structures transplantation group (n=13, groupB),pancreatic islets(200±50)and pancreatic islet-like structures (2×106)were transplanted under the kidney capsul in each rat;Diabetic control group(n=10, groupC), rats were implanted with culture medium without cells. The level of fasting blood glucose. of each rat was measured before transplantation and at 48 hours after the transplantation and dayly afterwards.Results:A large number of pancreatic stem cells were harvested after the digestion with the collegenase and purification with non-continuous density gradient. At day 1, few pancreatic stem cells were adhere to the bottom in the culture flask. Pancreatic stem cells were then proliferated and expanded rapidly within 5 days. At day 7 cobblestone like cell clusters were observed and expanded even bigger into the monolayer . Few islet-like buds appeared in the central or peripheral part of the monolayer. Thereafter more three dimensional Dithizon(DTZ) positive staining islet-like clusters were observed under the light microscope. A large number of islet-like clusters appeard from day 21 to day 27. Under the electronic microscope, from day 7 of culture, short stubby microvilli and apical junctional complexes were seen in most of cells. At day 15, short stubby microvilli in some cells disappeared while some interfilaments were presented around the granules of differrent densisties. In most of cells, interfilaments disappeared and some cells had high and some had lower density granules afterwords. Many granules containing cells were connected together to become a big cluster. Immunohistochemistry: at day 1, cells in the six-chamber slide showed strong positive CK-19 antibody staining. Cells were also positive of PDX-1 antibody staining. But these cells stained at different level even in a single cluster. There were islet-like buds appeared from 7 on. Most of cell clusters and islet-like buds were strongly positive CK-19 stained. More islet-like buds formed and growthed bigger from day 14 to day 27. They were stained strongly positive with PDX-1 antibody. The blood glucose of three groups all above 16.7mmol/L before transplantation. The blood glucose of rats in group A and group B decreased at 1 week after the transplantation. The decreased amplitude in groupB was larger than groupA,but two groups'blood glucose were still aboved 10mmol/L. In second week after the transplantation the blood glucose of rats in two groups decreased under 10mmol/L. The blood glucose in groupB was all lower than groupA.In third week after the transplantation,the Ievel of blood glucose greatly increased in groupA,However, The blood glucose level of groupB kept in 5mmol/L. The blood glucose in groupC was right in 16mmol/L,No one decreased.Conclusion:A large number of fresh islets and duct epithelial cells were harvested after the isolation and purification. The duct epithelial cells are potential stem cells which can be differentiated into insulin-producing cells at certain condition in vitro. Pancreatic islets and followed by pancreatic stem cell derived Islet-like structures Transplantation has a potential application for diabetes therapy.