Unequal Combinations Of Pdx1?Ngn3?Isl1?Nkx6.1 And Pax4 Induce The Differentiation Of Canine ADSCs Into IPCs

Diabetes is an important disease that threatens the health of humans and companion animals.At present,in addition to insulin control and drugs,islet transplantation is the most effective clinical treatment for diabetes.However,islet transplantation donors are scarce and immune rejection is serious.It is urgent to explore new methods of diabetes treatment.Inducing differentiation of pluripotent stem cells into Insulin producing cells(IPCs),which can replace damaged islet ? cells from the root,is expected to become a new idea for diabetes treatment.Adipose-derived mesenchymal stem cells(ADSCs)are easy to separate and have no ethical issues.Therefore,in this study,canine ADSCs were selected as transfection targets.On the basis of Pdx1,Ngn3,Pax4,Nkx6.1,the important regulatory genes for islet ?-cell development and function,the functionally verified Isl1 gene was introduced to form different multi-gene combinations.Adenovirus-mediated transfection induced canine ADSCs to explore Its effect on the differentiation of canine ADSCs into IPCs,the best combination was obtained by comparing the induction effects.The main findings are as follows:1.Comparison shows that adenovirus is significantly better than eukaryotic expression vectors in terms of transfection efficiency.Isl1 is the gene with the highest efficiency in inducing canine ADSCs to differentiate into IPCs among the three genes Isl1,Sox8 and HADH.2.Amplify the target gene fragment with the primer with homology arm and the synthesized target gene template,and successfully construct a multi-gene co-expression adenovirus vector using homologous recombination cloning technology: A combination p Ad Track-Pdx1-Ngn3-Isl1-Ad Easy,B combination p Ad Track-Pdx1-Ngn3-Pax4-Isl1-Ad Easy,C combination p Ad Track-Ngn3-Nkx6.1-Isl1-Pdx1-Ad Easy,D combination p Ad Track-Ngn3-Nkx6.1-Pax4-Pdx1-Ad Easy,E combination p Ad Track-Pdx1-Ngn3-Ad Easy,the vector was successfully packaged in 293 A cells to obtain infectious high-titer adenovirus venom,4 days after infection of dog ADSCs,different target genes of each combination were detected at different levels at the m RNA and protein levels expression.3.After 25 days of infection of canine ADSCs with multi-gene co-expression of adenovirus venom,the pancreatic islet-like cell group dithizone staining of A,B,and E combination showed scarlet,positive indirect immunofluorescence staining for insulin;The induction efficiency was the highest,and the detected 13 gene expression levels were upregulated relative to the Negative group.Under high glucose stimulation,the insulin secretion level detected in the supernatant was 91.26 ± 10.59 ?IU / 105 cells,and the cell lysate was detected.The insulin and proinsulin content was 99.82 ± 11.28 ?IU / 105 cells,and the insulin-stimulated release indexes in the supernatant and cell lysate were 2.07 ± 0.24 and 2.04 ± 0.35,respectively.In summary,in this study,adenovirus-mediated overexpression of different target gene combinations composed of Pdx1,Ngn3,Pax4,Nkx6.1,Isl1 in canine ADSCs.Among the combinations of four genes,the B combination Pdx1 + Ngn3 + Isl1 + Pax4 with the highest induction efficiency was selected,which provided new research references for the differentiation of genetically induced stem cells into IPCs,provided new methods for the clinical treatment of animal diabetes,and provided more research for the treatment of human diabetes data.