The Research Of Pancreatic Ductal Epithelial Cells Differentiate Into Pancreatic Endocrinal Cells.

Objective: To isolate and purify pancreatic ductal epithelial cells, then, proliferate and differentiate them into pancreatic islet cells.to test their insulin and C-peptide release under glucose compared with the normal islet cells,make preparation for the clinical application of pancreatic stem cells for treat diabetes mellitus. Substitute cell resource for the clinical application of islet transplantation,to improve stem cells cultivation, offer large cells source for the future stem cells biotechnology. Methods: pancreatic ductal epithelial cells were isolated and purified,then were identified with ck-19 by immunocytochemical stain,PDX-1 insulin by reverse transcriptase-polymerase chain reaction(RT-PCR), At the stage of 80-90% confluency,Pancreatic ductal epithelial cell cultures were set in serum-free medium which included exendin-4 KGF NIC. Monolayers of epithelial cells in culture gave rise to islet-like clusters within four weeks.The identity of neoislets was confirmed by dithizone staining and analysis of the gene expression for endocrine markers (Glut-2 PDX-1 insulin glucagon) by RT-PCR.then, the islet population' s function such as insulin C-peptide secretion in response to glucose in high(16.7mM) or low(3.3mM) level was researched by radioimmunoassay (RIA) and compared with the normal islet cells which were freshly extracted. Result: (l)After isolation and purification ,the pancreatic ductal epithelial cells could be proliferated efficiently, most cells expressed ck-19.(2) After four weeks,there was movement of the epithelial cells into three-dimensional cystic structures that DTZ staining was positive. The cellular extracts from neoislets expressed Glut-2 PDX-1 insulin, glucagon genes. (3) The three-dimensional cystic structures could release insulin and C-peptide in response to glucose as the normal islet could do,in detail, the three-dimensional cystic structures released insulin in high glucose concentration six times more than in the low. The neoislets secreted insulin which was 1/30 of the normal in low glucose and 1/35 in the high..they also secreted C-peptide under the same stimulation which was 1/32 of the normal in low glucose and 1/26 in the high. Conclusion:We have been able to expand rat duct epithelial cells efficiently and...