The Role Of SerpinB1 In Porcine Pancreatic Stem Cells Proliferation And Differentiation To Insulin Secretion Cells

Diabetes is a metabolic disease,also known as hyperglycemia,which is characterized by overeating,overdrinking and wasting.Long-term hyperglycemia can cause damage to tissues and organs,causing dysfunction in blood vessels,nerves,eyes,kidneys and heart.The functional impairment of islet β cells is the crux of diabetes.Pig pancreas stem cells(pPSCs)separated from pig pancreas,which was immortalized cell lines turned into T carrie.And the cell can be induced to insulin-producing cells.The human genome and the structure of insulin are similar to pigs,so it is important to study the mechanism of pPSCs proliferation.SerpinB1 is a serine protease inhibitor in neutrophils,which can alleviate tissue damage caused by inflammatory response by transplantation of protease activity.Studies have shown that when the liver is insulin resistance,it can promote the secretion of SerpinB1,which promotes the proliferation of pancreatic beta cells in the pancreas,and this reaction is related to the activity of the protease inhibitor.The self-renewal and differentiation of stem cells are regulated by various cytokines in the body.The effect of Serpin B1 on pancreatic stem cells as an adult stem cell is still unknown.This study has investigated the effect of SerpinB1 in pPSCs.We constructed SerpinB1 overexpression and interference cell lines.On the one hand,we confirmed that the over-expression SerpinB1 can promote the proliferation of pPSCs,on the other hand we found that SerpinB1 played an important role on the pPSCs differentiation.This study provides a theoretical basis for the proliferation mechanism of pPSCs,and provides a new method for the efficient acquisition of insulin secretion cells in vitro.1.SerpinB1 promoted the proliferation of pPSCs First,we detected the mRNA level of Serpin B1 in pPSCs by using qRT-PCR to,SerpinB1 was expressed in a large number of pPSCs.On the one hand,the expression of SerpinB1 in Sh-SerpinB1 cells decreased and increased in Ov-SerpinB1 cells.Through CCK-8 detection,it was found that interference with SerpinB1 decreased the cells activity,and SerpinB1 over-expression enhanced cell activity.The study of EdU,cycle testing and qRT-PCR results showed that compared with normal cells,EdU positive rate and S phase cell ratio of Sh-SerpinB1 cells lower than Ov-SerpinB1 cells.The mRNA expression of PCNA、CDK2 and CyclinD1 was lower in the Sh-SerpinB1 cells.These results suggested that SerpinB1 promote the cell cycle and promote pPSCs proliferation.At the same time,the results of Western blotting showed that over-expression of SerpinB1 activated the expression of STAT3,CDK2,and CyclinD1,inhibited the expression of P53 and P21.Therefore,we speculated that SerpinB1 promotes the proliferation of pPSCs through the STAT3 signaling pathway.2.SerpinB1 is essential for the pPSCs differentiation into insulin secretion cells.First,we used two steps induce system induce pPSCs to insulin-producing cells,q RT-PCR results showed that SerpinB1 expression in insulin-producing cells was higher than pPSCs.Then respectively detected the expression of SerpinB1 in the induction of 1d,3d,7d and 10 d sampling by Western blotting,and found that SerpinB1 expression increased in the whole process of induction.We induced respectively Sh-SerpinB1 and Ov-Serpin B1 cells,found that the cell masses from Sh-SerpinB1 were not dense,and were not easy to stick the wall,DTZ dyeing was negative.And the expression of Insulin,NeuroD1,PDX1,Glut-2,Nkx6.1 and MafA in Sh-SerpinB1 were lower than pPSCs.ELISA testing showed that Sh-SerpinB1 secreted lower Insulin and insensitive to high glucose.The cells from Ov-SerpinB1 performed better than the control.Therefore,we hypothesized that SerpinB1 affected the induction and differentiation efficiency of pPSCs to insulin-secreting cells.