Isolation and differentiation of pancreatic ductai epithelial cells of rats in vitro / 中华器官移植杂志

ABSTRACT

Objective To isolate and purify pancreatic ductal epithelial cells in adult rats,and induce differentiation of pancreatic ductal epithelial cells to islets in vitro.Methods By retrograde in- jection of collagenase into biliary-pancreatic tract,pancreatic tissues were digested and different types of pancreatic cells including islets,duct and degranulated aicni cells were separated by means of density gradient centrifugation.Ductal cells were purified by adhering method and identified by immunocyto- chemistry stain of ductal epithelial cells maker antigen(Cytokeratin 19,CK-19).Ductal cells were ex- panded in RMPI 1640 with 10% FBS.About one week when most adherent ceils were of monolayer, the medium was changed to serum-free DMEM/F12 supplemented with keratinocyte growth factor (KGF)to further expand ductal epithelial cells.When ceils reached 80% confluence,nicotinamide and high concentration of glucose were added to promote differentiation of pancreatic ductal epithelial cells.Islets like-structure was stained by Dithizone.Results Irnmunocytochemistry stain of CK-19 re- vealed that most isolated ceils were ductal epithelial ceils.The cultured ductal epithelial cells began to adhere at day 1,reached 80% confluence and cell clones were formed at day 14-21.At day 28,islets- like-structure appeared and was positive for Dithizone staining.Conclusions Ductal epithelial cells of rats can be isolated by means of density gradient centrifugation and purified by adhering method.Duc- tal epithelial cells can differentiate into islets-like-structure in vitro.