| ObjectiveType 1 diabetes (autoimmune ) is a disease due to an inadequate mass of functional pancreatic cells. Current treatment is insulin injection, which can't cure diabetes, but also bring suffering to patients. A new strategy is -cell replacement therapy via islet transplantation. A recent report by Shapiro et al has demonstrated that using a glucocorticoid-free immunosuppressive therapy combind 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 seven type 1 diabetic patiens. However, such an approach is limited by the scarity of the transplantation material 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 impaired cells of diabetic patients. Stem cells are defined as clonogenic cells capable of both self-renewal and multilineage differentiation . There is clear evidence that stem cells committed to differentiate into insulin-secreting cells, serving as a possible future source for cells replacement therapy in diabetes. In order to look for suitablecells-replacement source and improve the technique of stem cells, pancreatic ductal epithelial cells from Kunming mice were separated, cultured and transdifferentiated into insulin-producing cells in this experiment. Materials and MethodsPancreas 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 microscopic examination and for immunocytochemical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19. The expression of insulin and glucagon gene were determined by RT-PCR. DTZ stain experiment was done to examine whether differentiated islet-like clusters have endocrine function. Study were performed to determine whether the new P cells in these islet-like clusters could secrete insulin in response to glucose. Results1. Primary culture of pancreatic ductal epithelial cells from Kunming mice: At the beginning of separation, typical epitheliod cells exist in single globular shape. These cells proliferated quickly when the medium had been changed to full-medium 48 hours later. These cells then were grown for about 1-2 weeks until reaching near confluence or forming substantial plaques of epithelial cells in cobblestone pattern. 2-3 weeks later, epitheliod cells gathered gradually and formed islet-like clusters.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-l+on the 16th day. It proved that pancreatic ductal epithelial cells could proliferate quickly by this way and have 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 very low levels of insulin and glucagon mRNA in the starting material but increase were found as islet-like clusters developed. The initial insulin mRNA levels of IOD was 0.363 + 0.038, whereas, from islet-like clusters at 16th day, it was 1.960 + 0.117;Similarly, glucagon mRNA levels increased from the initial separating cells being 0.273 0.042 and harvested islet-like clusters being 1.644 0.090, demonstrating pancreatic stem cells differentiating into insulin-expressing cells.4.DTZ stainng: On the first day, there were seldom cells DTZ stain positive, while at the 16th day developed islet-like clusters stain red. It illustrated islet-like cells have the ability to produce insulin.5.Glucose stimulation...
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