| Diabetes mellitus is a group of metabolic diseases,which is threatening many people's health.Transplantation of islets of Langerhans offers an attractive strategy for diabetes therapies,but its wide application has been limited by donor shortage and immunological rejection after transplantation.Adult pancreatic stem cells with strong proliferatve capacity and multipotential may be potential cell source since they have no ethical issues and teratoma formation.But researches on adult pancreatic stem cell progress slowly because of lacking recognized specific markers and credible detecting systems.So,the major task is localizing and identifying adult pancreatic stem cells.In this research,different dosages of BrdU were injected subcutaneously to neonatal mice for 6 times or interperitoneally to pregnant 14th day mice for 5 to 6 times,by which we tried to find out the best method of labeling mouse pancreatic cells with high efficiency;then,the neonatal mice born by pregnant mice injected interperitoneally five times of BrdU were long time traced to make certain the localization of label-retaining cells(LRCs) in pancreas in vivo; The diabetic BrdU-chasing mice induced by streptozotocin were used to explored the LRCs′proliferative capacity and multipotential,especially their potential of differentiation intoβcells;LRCs′proliferative capacity in vitro was explored by three kinds of culturing methods, including in vitro-G418 treatment,classifying inoculation of pancreatic single cells got from complete digestion or multistep digestion of pancreas.The results showed that injecting interperitoneally pregnant 14th day mice for 5 times is the best method of labeling mouse pancreas high efficiently;By this method,after the neonatal mice were chasing for 10 weeks,the LRCs were found in the islets and around acini,but not in big ducts;the LRCs in mouse pancreas can proliferate after STZ administration for 7 days and differentiate into insulin-secreting cells after STZ administration for 10 days;in the in vitro culture tests,50μg/mL G418 added in culture medium for 48h could wipe off fibroblasts and purify epithelial cells,but the left epithelial cells were hard to proliferate into single cell clusters;the method of classifying inoculating of pancreatic single cells got from complete digestion of pancreas could reach the aim of removing fibroblasts and purifying epithelial cells, but couldn't achieve the aim of screening out adult pancreatic stem/progenitor cells;In the third strategy,the pancreatic single cell suspensions after digestion for 20min were inoculated and culture for the first 40min,the unattached cells were moved to another plate and culture for 24 hours,then the unattached cells were moved to a new plate and culture for another 24 hours, at last the upper unattached cells were discarded.All the attached cells in the foregoing steps were cultured with medium containing 2%FBS for 7 to 10 days.By this method,we screened out a group of cells with strong proliferative capacity and cluster formation capability from the first and second 24 hours attached cells,these ceils were demonstrated to be candidate pancreatic stem/progenitor cells by RT-PCR and immuno-stainning analysis.And also some cells were proved deriving from LRCs with immuno-stainning detection.The in vivo and in vitro experiment results suggested that LRCs were a group of adult pancreatic stem/progenitor cells in adult mouse pancreas.These results may help for further exploring the specific markers of pancreatic stem cells,mechanism of their proliferation and directional differentiation,,and also preparing the conditions for applications of adult stem cells in human clinic therapy of pancreatic diseases in future.
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