The Effects Of Folic Acid On The Proliferation And Differentiation Into Insulin-secreting Cells Of Porcine Pancreatic Stem Cells

Diabetes mellitus(DM)is a type of chronic metabolic disease that poses an extremely serious threat to public health due to its high morbidity and high fatality rates with multiple complications.The main cause of DM is the lack of insulin secretion or utilization disorders caused by the damage of pancreatic ?-cells.At present,many studies on DM treatment have focused on cell therapy.Porcine pancreatic stem cells(pPSCs)are a type of stem cells isolated from fetal porcine pancreas in our laboratory which can be induced into insulin-producing cells(IPCs)in vitro.In addition,the structure and size of porcine islets are most similar to humans,so IPCs induced by pPSCs are expected to be used for clinical treatment of human DM.Folic acid(FA)has long been considered to participate in DNA and RNA synthesis processes as vitamins.FA can also affect the gene expression level in vivo through epigenetic regulation,and ultimately affect the self-renewal,proliferation,and differentiation of cells,as a mediator of carbon units.However,recent studies show for the first time that FA can directly promote the proliferation of germline stem cells as a signaling molecule.So far,the effect and mechanism of FA on the proliferation and differentiation of pPSCs remains unclear.This experiment explored the roles of FA on the proliferation and differentiation into IPCs of pPSCs,and initially involved the mechanisms of these effects,and gained the following results:1.FA could promote the proliferation of pPSCs through activating the Wnt signaling pathway and ERK pathway mediated by FOLR?Firstly,the proliferative effects of FA on pPSCs were evaluated by CCK8 and EdU.We found that the EdU positive rate of FA-treated cells were higher tahn control cells,I ndicating that FA promoted the proliferation of pPSCs.Next,the results of qRT-PCR and Western blotting experiments showed that the proliferation associated genes PCNA,CyclinD1,and c-Myc in the pPSCs were up-regulated both in protein and RNA levels after FA treatment,demonstrating that FA accelerated the proliferation of pPSCs.With the addition of the FA inhibitor MTX,these indicators were all decreased,further clarifying the pro-proliferation effect of FA on pPSCs.At the same time,we found that with the up-regulation of proliferation of pPSCs,FA stimulated the expression of folic acid receptor ?(FOLR?).Interfering with expression of FOLR? inhibited the proliferation of pPSCs,indicating that FA promotes the proliferation of pPSCs through binding to the FOLR?.In mechanism studies,Western blotting showed that the expression of active ?-catenin and p-ERK,the key proteins in the classical Wnt signaling pathway and ERK signaling pathway,increased in the pPSCs treated with FA.After further adding the two pathway's inhibitors respectively,the pro-proliferation effect of FA was also significantly inhibited.The above results indicate that FA could activate proliferation of pPSCs through activating the Wnt and ERK pathway mediated by FOLR?.2.FA improved the induction efficiency of pPSCs to IPCsOur previous studies have confirmed that pPSCs can be induced into IPCs in vitro.Based on this,I added the FA to the original culture system to explore the effect of pPSCs induced to IPCs.The results showed that in the cell cluster obtained by adding FA during the induction,the positive rate of DTZ and the expression levels of the functional proteins Insulin and Cpeptide were increased,and the ? cell maturation markers Insulin,NKX6.1,MafA,and NeuroD1 were up-regulated at the mRNA level.And most importantly the ability to secrete insulin in response to high glucose for the cells increased,compared to the normal induction group.In addition,similar to proliferation experiments,Western blotting showed that the expression of active ?-catenin and p-ERK were up-regulated in cell clusters obtained by adding FA during induction.Further,qRT-PCR showed that when the two pathways were inhibited during induction,the expression of Insulin,NKX6.1,MafA,and NeuroD1 were down-regulated correspondingly,indicating that the pro-differentiation effect of FA on pPSCs was inhibited.These results indicated that the effect of FA on the process of differentiation might be regulated by Wnt and ERK signaling pathway.In conclusion,in this study,we confirmed that FA could promote the proliferation of pPSCs mediated by FOLR? and the differentiation into insulin producing cells in vitro;and these effects of FA on pPSCs could be regulated by Wnt and ERK signaling pathway.