Isolation,Cultivation And Identification Of Pancreatic Stem-Like Cells Of Newly Born Kunming Mouses

Objective: This study was designed to isolate ,cultivate and identify pancreatic stem-like cells from newly born Kunming mouses'pancreas.Under the condition of feeder layer consisted of fetal fibroblasts , pancreatic stem-like cells were desired to subculture continuously,so that we could obtain a group of pancreatic stem-like cells ,which had high unicity and quantity.Methods: The pancreas were digested by collagenase ofⅤtype,endocrine portion'cells and exocrine portion'cells were separated by Percoll discontinuous density gradient.All of the cells were cultured with serum-free DMEM medium added with bFGF and EGF,and observed by invert phase contrast microscope.Pancreatic stem-like cells were identified by immunocytochemistry staining using antibodies against Nestin,and were subcultured under the condition of feeder layer,which were fetal fibroblasts effected by mytomycin C.Result:Pancreatic endocrine portion'cells and exocrine portion'cells were separated by Percoll discontinuous density gradient ,so that they located in different density interfaces.There were Nestin-positive pancreatic stem-like cells in both pancreatic endocrine and exocrine portions,which were undifferentiated and highly reproductive.With the cultural time extension,pancreatic stem-like cells derived from endocrine portion,had the tendency to differentiate intoβ-cells;pancreatic stem-like cells derived from exocrine portion,had the tendency to differentiate into pancreatic duct epithelial cells.Pancreatic stem-like cells could subculture...