The Differentiation Of Canine ADSCs Into IPCs Via The Co-transfection Of Pdx1/Ngn3/Mnx1/Mafa/Pax4

At present,by re-programing pancreatic development-related genes,adipose-derived mesenchymal stem cells can have insulin secretion function,thus replacing pancreatic islet?cells for diabetes treatment has become a research hotspot.The genes Pdx1,Ngn3,Pax4,Mafa,etc.are the main regulatory genes of pancreas development,and their mutations and deletions will seriously damage the development of pancreatic endocrine cells.These genes were overexpressed in stem cells by transgenic technology,but the results showed that the single-gene transfection did not have a significant induction effect.To achieve the directed differentiation of stem cells into insulin-positive cells,other genes were needed.It is of great significance to screen the best gene combination and achieve differentiation of stem cells into insulin-secreting cells through multi-gene co-expression.In the early stage,the research team has screened the new expression regulation genes Foxa2,Insm1 and Mnx1 which may play a role in the differentiation of canine ADSCs into insulin secreting positive cells through absolute quantitative transcriptome sequencing analysis.Therefore,in the early stage of this experiment,eukaryotic overexpression vectors and adenovirus overexpression vectors linked with genes Foxa2,Insm1,and Mnx1 were constructed.The over-expression function in canine ADSCs was verified,and finally the new expression-regulated gene Mnx1with the best function was selected.Besides,the test found that eukaryotic expression vectors have a long screening period and low screening efficiency,while adenovirus overexpression vectors are simple to operate and have high infection efficiency.Therefore,in the later experiments,the gene Mnx1 and the key genes Pdx1,Ngn3,Pax4,and Mafa were linked by 2A peptides,and five sets of adenovirus-mediated multi-gene co-expression vectors were constructed by different arrangements.The adenovirus venom expanded into high titers through packaging was used to infect canine adipose-derived mesenchymal stem cells and monitor the differentiation effect.The research results are as follows:1. Successfully constructed recombinant eukaryotic overexpression vectors pIRES2-EGFP-Foxa2,pIRES2-EGFP-Insm1,pIRES2-EGFP-Mnx1 and recombinant adenovirus overexpression vectors pAd-Foxa2,pAd-Insm1,pAd-Mnx1,and identified by PCR and double enzyme digestion.Comparing the m RNA expression of islet beta cell develo pment-related genes in each time period after infection of canine ADSCs,it was sho wn that adenovirus-mediated gene Mnx1 overexpression can significantly increase the expression levels of islet beta cell development-related genes Pdx1,Ngn3,Mnx1,Nkx2.2,Nkx6.1,Gata4 in canine ADSCs.It was also found that the infection efficiency of adenovirus was significantly higher than that of eukaryotic overexpression vector.2. Successfully constructed five sets of multi-gene co-expression adenovirus vectors pAd-Pdx1-E2A-Ngn3,pAd-Pdx1-E2A-Ngn3-P2A-Mnx1,pAd-Pdx1-E2A-Ngn3-P2A-Mafa-F2A-Mnx1,pAd-Pdx1-E2A-Ngn3-P2A-Pax4-E2A-Mnx1,pAd-Pdx1-E2A-Ngn3-P2A-Mafa-F2A-Pax4.Digestion,PCR identification and sequencing are correct.Through RT-qPCR and Western blot detection,it was found that the target gene in each combination was significantly expressed at the mRNA level and protein level.3. Five sets of multi-gene co-expression adenovirus vectors infected canine ADSCs after packaging amplification.Fluorescence quantitative PCR results showed that after the co-expression of genes Pdx1,Ngn3,Mafa and Mnx1,the expression levels of pancreatic related genes Pdx1,Mafa,Nkx2.2,Nkx6.1,Gata4,Ins,Pcsk2,Slc30a8,Abcc8,Ksnj8,G6pc2 were significantly increased.Compared with other combinations,the difference is significant.Dithizone staining was performed on the 25th day after infection,and it was found that the formed round cell-like clusters appeared reddish brown.This suggests that there is insulin secretion.Adenovirus-infected canine ADSCs of combination three were stimulated by high glucose(25 mmol/L)and low glucose(5 mmol/L),the insulin secretion in the supernatant was 101.810±4.39?IU/10 ~5cells and 49.439±2.5?IU/10 ~5cells.The insulin stimulation index(SI)is 2.059±0.175,which is significantly different from other groups.The results showed that overexpression of gene Mnx1 can significantly increase the expression levels of pancreatic islet?cell development regulation genes Pdx1,Ngn3,Mnx1,Nkx2.2,Nkx6.1,and Gata4 in canine ADSCs.The infection efficiency of adenovirus is significantly higher than that of eukaryotic overexpression vector.Adeno virus-mediated genes Pdx1,Ngn3,Mafa and Mnx1 are co-expressed in canine adipose-derived mesenchymal stem cells,which can efficiently regulate their differentiation into insulin-producing positive cells.It has laid the foundation for further exploration of stem cell replacement therapy for diabetes.