The Differentiation Mechanism Of BMSCs Into Insulin-secreting Cells In Injured Pancreatic Extract

Part I:The effect of the injured rat pancreatic extract on transdifferentiation of BMSCs into insulin-secreting cellsObjective:To explore the effect and the mechanism of injured pancreatic extract from Sprague-Dawley rats on induction of pancreatic differentiation of BMSCs through continuous dynamic monitoring of gene, protein, cell morphological changes.Methods:About 90% partial pancreatectomy was performed with six-week-old rats. injured pancreatic extract derived from the remnant pancreas after 48 h. After the BMSCs were induced with injured pancreatic extract for 14 days in vitro, morphological changes of the induced cells were observed. Immunocytochemical staining were used to examine pancreatic developmental protein. RT-PCR and real-time PCR were conducted to detect the expression of pancreatic endocrine gene, and Flow cytometry was used to detect the efficiency of transdifferentiation. ELISA were performed to evaluate the function of the IPCs.Results:Insulin-secreting cells can be transdifferentiated from BMSCs by the inducement of injured pancreatic extract. during the process of induction, BMSCs can express a series of genes closely related to pancreatic development, such as PDX-1, NKX6.1, GLUT2, PC2, C-peptide, etc. and also express the protein expression of mature pancreas, such as:C-peptide, Insulin, Nkx6.1 etc. the differentiated BMSCs cells obtained nearly 13.3±1.8% insulin-positive cells by flow cytometry analysis and released 1/20-1/50 insulin in response to glucose stimuli comparable to that of natual islets.Conclusion:affter pancreatic injured,injured pancreatic extract is rich in pancreatic development associated protein,which can induce transdifferentiation of BMSCs to insulin-secreting cells. Part II:Screening the related protein induced differentiation from injured pancreatic extract by mass spectrometryObjective:Injured pancreatic extract contain associated protein which can induced transdifferentiation of BMSCs into insulin-secreting cells.by using mass spectrometry. To filter out soluble proteins from injured rats pancreatic extract and normal rats pancreatic extract that can highly efficiently induced BMSCs to differentiate into insulin-secreting cells in vitro.Methods:differential protein profile betwent injured rats pancreatic extract and normal rats pancreatic extract were analyed by using two-dimensional gel electrophoresis (2-DE). The gels were visualized by silver staining and analysed with Image Master 2D Elite software.The protein spots with signifieant changes were visualized by Coomassie blue staining for further identifieation. Afterwards, the differential proteins were identified via in-gel digestion combined with MALDI-TOF-TOF-MS. Cofilin-1,NDPKA,PRDX6 and HTRA2 were analyzed in rat injured pancreatic extract with Western blot to confirm the resμlts of 2-DE.Resμlts:The average spots in gels for injured rats pancreatic extract and normal rats pancreatic extract were 1227±17 and1507±29, respectively.50 spots displayed at least 2-fold of differential expression. and 20 proteins were successfμlly identified by MS,7 spots(7,15,16,23,34,35,42) were up-regμlated, and 13 spots were down-regμlated. cofilin-1,NDPKA,HINT1,PPIB,PRDX6 and HTRA2 are associated with cells differentiation and proliferation. These protein were detected from injured rats pancreatic extract by Western blot.Conclusions:Injured rats pancreatic extract contain pancreatic development associated protein. By MS,we found injured rats pancreatic extract contains cofilin-1,NDPKA,HINT1,PPIB,PRDX6 and HTRA2 which can promote transdifferentiation of BMSCs to insulin-secreting cells. They can provide new factor to highly efficiently induced cell differentiation.