| Objectives Patients with type 1 diabetes must be treated with insulin throughout life, and the treatment can't prevent complications completely .So it is very important to find a new method replacingβ-cell function in vivo. A recent report by Shapiro has demonstrated that using a glucocorticoid-free immunosuppressive therapy combined with the infusion of an adequate fresh islet mass resulted in insulin independence and good metabolic control for periods of more than 12 months in 7 patients with type 1 diabetes. However, such an approach is limited by the scarcity of the transplant and the long-term side effects of immunosuppressive therapy. These problems may be overcome by using a renewable source of cells, such as islet-cells derived from stem cells, which provide us a new strategy to treatment of diabetes: transdifferentiate stem cells into islet cells in vitro in order to replace disfunctioning βcells of diabetic patients. There is clear evidence that stem cells committed to differentiate into insulin-secreting cells, serve as a possible future source for cell- replacement therapy in diabetes. In order to look for suitable cell-replacement source and improve the technique of stem cells, pancreatic ductal epithelial cells from Kunming mice were separated, cultured, transdifferentiated into insulin-producing cells and tansplanted into abdominal cavity of mice in the experiment. This led us to investigate whether differentiated islet-like clusters could be stimulated to produce insulin in vivo. Materials and Methods Pancreas of Kunming mice were digested with collagenase type Ⅴ, followed by filtrating to separate pancreatic ductal epithelial cells from islets and acinar tissue. Ductal epithelial cells were cultivated in DMEM/F12 medium with the addition of growth factors. Samples were taken at different time points for light and electronic microscopic examination and for immunocytochemical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19. The expression of insulin and glucagon genes was determined by RT-PCR. DTZ stain experiments were done to examine whether differentiated islet-like clusters had the function of producing insulin. In order to investigate whether differentiated islet-like clusters could be stimulated to differentiate into functional islets if placed in vivo environment, islet-like clusters transplantation was performed. Model mice of type 1diabetes made by streptozotocin injection into abdominal cavity must have high blood glucose levels over 16.6mmol/L twice a days, The diabetic mice were divided into two groups (n=6) ad libitum, 6 diabetic mice in each group, and there were 6 normal mice as control (n=6). Group 1 was experimental mice implanted with islets about 3500 equivalent into abdominal cavity, group 2 was implanted same volume of normal saline. Group 3 was normal control implanted same volume of normal saline. All mice were examined for blood glucose levels and observed for life conditions regularly. Experiment period was 2 months. Results 1. Primary culture of pancreatic ductal epithelial cells from Kunmingmice: At the beginning of separation, typical epithelioid cells existed in single globular shape. These cells proliferated quickly when the medium had been changed to full-medium 48 hours later. These cells then grew for about 1-2 weeks until reaching near confluence or forming substantial plaques of epithelial cells in cobblestone pattern, epithelioid cells gathered gradually and formed islet-like clusters 2-3 weeks later. 2. Immunocytochemistry: At the beginning of isolation a large number of epithelioid cells were CK-19 immunoreactive positive and few of them were PDX-1 positive, while the number of CK-19+ cells increased significantly and most cells were PDX-1+ on the 16th day. It proved that pancreatic ductal epithelial cells could proliferate quickly by this way and had the potency of transdifferentiation into pancreatic stem cells. 3. RT-PCR inspecting the expression of insulin and glucagon: The analysis of mRNA by RT-PCR showed ve...
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