Study On Reprogramming Of Canine Adipose Mesenchymal Stem Cells To Insulin Secreting Cells By FoxA1、Pdx1、Ngn3 And Pax4/Nkx2.2

Diabetes is a metabolic disease characterized by hyperglycemia.At present,the clinical treatment of diabetes is mainly based on injecting exogenous insulin,but this method cannot maintain normal blood glucose levels for a long time,and the risk of complications still exists.Islet transplantation is an effective method to solve this problem,but there are problems such as insufficient donors and immune rejection.Therefore,it has become a research hotspot to efficiently produce insulin-secreting cells(IPCs)in vitro instead of islet transplantation.Adipose-derived mesenchymal stem cells(ADSCs)are ideal seed cells for IPCs due to their wide source,easy access to materials,low immunogenicity,multi-differentiation.Studies have shown that ADSCs can differentiate into IPCs under in vitro induction conditions,but they have some shortcomings such as low differentiation efficiency and low insulin secretion.In our previous work,FoxA1,a novel differentially expressed gene that induces the differentiation of canine ADSCs into IPCs in vitro,was screened out by absolute quantitative transcriptome sequencing.In order to verify the influence of FoxA1 gene on the differentiation of canine ADSCs to IPCs in vitro,we obtained a FoxA1 overexpressed ADSCs cell line by constructing a FoxA1 recombinant adenovirus expression vector which was inoculated in 293 A cells.After that,we used the procedure of inducing the differentiation of canine ADSCs to IPCs in vitro to explore the influence of FoxA1 gene overexpression on canine ADSCs’ differentiation IPCs in vitro.We obtained a FoxA1 gene interference ADSCs cell line by constructing a FoxA1 gene siRNA vector to transfect canine ADSCs.Then we used the procedure of inducing the differentiation of canine ADSCs to IPCs in vitro to explore the influence of FoxA1 gene interference on canine ADSCs’ differentiation IPCs in vitro.The key genes of the pancreatic developmental cascade regulation(Pdx1,Ngn3,Pax4,and Nkx2.2)can be transfected into ADSCs alone or in combination to induce differentiation into IPCs,but the differentiation efficiency is low,and the IPCs formed by differentiation are immature.To further improve the differentiation efficiency of ADSCs to IPCs and the maturity of IPCs,in this study,FoxA1 Pdx1,Ngn3,Pax4,Nkx2.2 genes were combined into different combinations: A.FoxA1+Pdx1+Ngn3,B.,FoxA1+Pdx1+Ngn3+Pax4 and C.FoxA1+Pdx1+Ngn3+Nkx2.2.Different 2A peptide sequences were added to each gene sequence and linked using homologous recombination technology.A multi-gene recombinant adenovirus vector was constructed and infected with canine ADSCs after infecting 293 A cells.The optimal combination for reprogramming canine ADSCs to IPCs was investigated only through continuous cell culture.The main results are as follows:1、The cells induced by FoxA1 gene overexpression and interference with canine ADSCs were subjected to glucose-stimulated insulin secretion tests and RT-qPCR to detect the expression of pancreatic β-cell-related genes.Compared with the simple induction group cells,in the FoxA1 gene overexpression group,the amount of insulin-stimulated by low glucose increased significantly(p<0.05),and the amount of insulin-stimulated by high glucose and KCl increased significantly(p<0.01),the expression levels of Pdx1,Mafa,Nkx6.1,Pax4,Pcsk1,Pcsk2 and Ins were significantly decreased(p<0.01).Therefore,the expression of FoxA1 gene promotes the differentiation of canine ADSCs into IPCs in vitro.2、The number of clumps,dithizone staining,glucose-stimulated insulin secretion test,and RT-qPCR to detect the expression of pancreatic β-cell-related genes were performed on the cells that were combined with each combination transfected with canine ADSCs to induce differentiation into IPCs.The number of clusters of combination B and combination C was significantly higher than that of combination A(p<0.05);the amount of low glucose,high glucose,and KCl stimulated insulin secretion in combination B was significantly higher than that of combination A and combination C(p<0.01);The expressions of Pdx1,Mafa,Nkx6.1,Pcsk2,Ins I genes of combination B and combination C were significantly higher than those of combination A(p<0.01).Therefore,FoxA1,Pdx1,Pax4,Ngn3 reprogrammed canine ADSCs to become IPCs with the highest efficiency,and differentiated IPCs are the most mature.In conclusion,the expression of the FoxA1 gene promotes the differentiation of canine ADSCs into IPCs in vitro.FoxA1,Pdx1,Pax4,Ngn3 can reprogram canine ADSCs into IPCs.