Preliminary Study Of Pancreatic Stem Cells Of Amplification Subculture In Situ And In Transplantated In Vivo

Objective:To optimizate and stabilize the conditions of culturing pancreatic stem cells in vitro; to explore efficient methods to obtain a higher enriched pancreatic stem cells; to detect the subsistence of pancreatic stem cells by transplantated in vivo.Methods:The pancreatic stem cells were isolated from the pancreases of newborn SPF kunming mice which digested with collagenas IV,by the methods of cell suspension culture and tissue culture.The pancreatic stem cells were cultured in Dulbecco modified eagle medium(DMEM) with 10%FBS during the first two days and serum-free medium soon after.Inverted microscope were used to observe the morphological characteristics an growth characteristics.The specific marker of pancreatic stem cells(Nestin) could be detected by immunohistochemical staining techniques and methods.A higher enriched pancreatic stem cells were got by amplification subculture in situ.The pancreatic stem cells of second generation marked with DAPI and Brdu were injested into leg muscle and lateral ventricles of kunming mice,and observe the natural subsistence of pancreatic stem cells in different time by immunohistochemical staining.Results:After culturing in vitro for 24h,the typical pancreatic fibro-blasts appeared,and they reached 70～80% confluence after one week tissue culture or 3～4 days cell suspension culture. The pancreatic stem cells with active proliferative capacity emerge at this time and express the specific marke(Nestin).The pancreatic stem cells with undifferentiated and strong proliferative ability could be cultured after repeatedly amplification subculture in situ.The amplified pancreatic stem cells of the second generation marked with DAPI were injected into the leg muscle,but could not be detected after one week.While the pancrestic stem cells marked with Brdu were injected into lateral ventricles,and could be detected after 3 days,and gradually decreased after one week,but could not be detected after two weeks.Conclusion:Firstly,we can obtain a higher enrichment of pancreatic stem cells by the methods of amplification subculture in situ. Secondly,pancreatic stem cells can suvival one week in lateral ventricle of mouse.