In Vitro Pancreas Duodenal Homeobox-1 Enhances The Differentiation Of Pancreatic Ductal Epithelial Cells Into Insulin-Producing Cells

Part I Primary Culture,Subculture and transfection of Rat Pancreatic Ductal Epithelial CellsObjective To establish the system of the primary culture and subculture of normal pancreatic ductal epithelial cells, then to transfect PDX-1 gene into pancreatic ductal epithelial cells in vitro for further study.Methods Rat pancreatic tissue was submitted to digestion by collegenase V, ductal epithelial cells were separated by density gradient centrifugation and then were cultured in RPMI1640 medium with 10%FBS. When the cultured cells reached 80%-90% confluence, they were subcultured in 1∶2. After 3～5 passages, they were incubated in six-well plate 24 h before transfection of recombination plasmids PDX-1(XlHbox8VP16). There were two groups. One group was the transfected group, another was the control group.Results Appearance of epithelial cells in growth way proved the cultures were pancreatic ductal epithelial cells. These pancreatic ductal epithelial cells had been subcultured for from three to five generations.Conclusion Our culture methods and conditions used in the experiments are suitable for the primary culture and subculture of rat pancreatic ductal epithelial cells. And we also had transfected PDX-1into rat pancreatic ductal epithelial cells successfully. PartⅡIn Vitro the Role of Pancreas Duodenal Homeobox-1 in the Differentiation of Pancreatic Ductal Epithelial Cells and Identification of Insulin-producing cellsObjective To transfect PDX-1 gene into pancreatic ductal epithelial cells in vitro and observe the role of Pancreas Duodenal Homeobox-1 in differentiation of pancreatic ductal epithelial cells into insulin-producing cells.Methods To culture the transfection and the control, then real-time RT-PCR was used to dectect the expression of PDX-1 and Insulin mRNA in the pancreatic ductal epithelial cells and Western blotting analyzed the expression of PDX-1 and Insulin protein at vary time. The insulin content of secretion was detected by RIA. The insulin-producing cells were detected by Dithizon-staining.Results XlHbox8 mRNA was expressed in pancreatic ductal epithelial cells. PDX-1 and insulin mRNA as well as PDX-1 and insulin protein were significantly increased in the transfected group. The production and insulin secretion of insulin-producing cells differentiated from pancreatic ductal epithelial cells were higher than those of the untransfected cells in vitro with a significant difference (1.32±0.43 vs 3.48±0.81, P < 0.01 at 5.6 mmol/L; 4.86±1.15 vs 10.25±1.32, P < 0.01 at 16.7 mmol/L).Conclusions PDX-1 can promote rat pancreatic ductal epithelial cells to differentiate into insulin-producing cells significantly in vitro. In vitro PDX-1 transfection is a valuable strategy for increasing the source of insulin-producing cells.