Preliminary Study Of Pancreatic Stem Cells Of Induction And Diffe-Retiation Into Pancreatic Islet-Like Structure In Vivo

Objective:To optimizate the method and technique of pancreatic stem cells amplification subculture in situ;to estabilish and obtain a stable pancreatic stem cells series;to obtain plenty of pancreatic stem cells for induction and differetiation into pancreatic islet-like structure in vivo.Methods:The pancreatic stem cells series were established by amplifying pancreatic stem cells with the optimized method of amplification subculture in situ.The secondary generation pancreatic stem cells with fine growth condition were seperated into four groups:control group,glucose group,nicotinamide group and serum nicotinamide group,after continuous induction and differetiation for 4 weeks,changes of morphological characteristics of the PSCs were observed under light microscope.After the procedure of induction,DTZ staining and Mallory staining were used to preliminarily identify the pancreatic islet-like struture;Immunofluorescence staining were used to detect whether insulin-positive cells were existed in the struture.Results:Control group:the morphological characteristics and growth patterns of pancreatic stem cells were changed after continuous culturing in vivo,from round,strong three-dimensional to flat,less refraction and weak three-dimensinal struture;glucose group:after 9 days induction,the pancreatic stem cells gathered and grown like string,after 20 days induction,the cells turned into flat shape;nicotinamide group:after 5 days induction,the cells started gathering together,and intended to form a cluster after 25 days induction;serum nicotinamide group:after one week induction,the pancreatic stem cells gathered together and form cell clusters,the cell clusters grown and became larger after two weeks induction,after three weeks induction,the pancreatic islet-like structures were formed with complete capsule.Brownish red color were observed with DTZ staining;Mallory staining identified two kinds of cells in the pancreatic islet-like struture:the surrounding cells present pale yellow and the center cells present red.Under the UV light excitation,the insulin-positive cells showed green fluorescence by insulin antibody immunofluorescence staining.Conclusions:(1) The pancreatic stem cells series were established after amplificated by the optimizated method of amplification subculture in situ.A higher enrichment of pancreatic stem cells can be obtained for further experiment;(2)Under the induction condition of serum and nicotinamide,the pancreatic stem cells could differetiate into pancreatic islet-like structure,with complete capsule,DTZ staining positive,Mallory staining present two kinds of cells and Immunofluorescence staining identify insulin-positive cells.