Study On Reprogramming Of Canine ADSCs Into IPCs By Transfection With Pdx1,Ngn3,Sox9,Pax4 And Nkx2.2 In Different Combinations

Diabetes is one of the most common metabolic diseases in the world.Traditional drug therapy and insulin injection therapy cannot fundamentally treat diabetes.Islet transplantation can effectively control blood glucose changes and avoid complications,but its application is limited due to donor deficiency and immune rejection.At the cellular level,pluripotent stem cells can be induced to differentiate into cells with insulin secretion function through targeted induction,which can provide a new therapy for the treatment of diabetes.Adipose-derived mesenchymal stem cells(ADSCs)are ideal sources for treatment of diabetes,because they are derived from adipose tissue,easy to isolate and culture,and involved fewer medical ethical issues.As key genes for cascade regulation,Pdx1,Ngn3,Sox9,Pax4,and Nkx2.2 play important regulatory roles in the development and maturation of pancreas and islet cells.Studies have shown that ADSCs can be induced to differentiate into insulin producing cells(IPCs)by transfection of these genes,but the insulin content secreted by IPCs does not change with the concentration of sugar and the IPCs do not have the properties of functional isletβcells.The multi-gene combination transfection method formed in cooperation with other genes can provide new ideas for the reprogramming of ADSCs.Therefore,this experiment introduced the functionally verified Sox9 gene on the basis of these cascade-regulated key genes,and explored the effects of five different multigene recombinant adenoviruses on the induction of canine ADSCs into IPCs.The main research results are as follows:1. The canine ADSCs were infected with Sox9,Fev,Gata4 single-gene eukaryotic overexpression vector and adenovirus overexpression vector that were successfully constructed,respectively.By comparing the morphology of the infected cells and the m RNA levels of genes related to islet development,we found that the transfection efficiency of the eukaryotic overexpression vector was too low to meet the test requirements.The adenovirus overexpression vector has better transfection efficiency and is an ideal transfection tool in gene therapy.And Sox9 gene overexpression adenovirus had the best induction effect.2. Five groups of multi-gene co-expression adenovirus vectors(A:p Ad Track-Pdx1-Ngn3-Ad Easy;B:p Ad Track-Pdx1-Ngn3-Sox9-Ad Easy;C:p Ad Track-Pdx1-Ngn3-Pax4-Sox9-Ad Easy;D:p Ad Track-Pdx1-Ngn3-Nkx2.2-Sox9-Ad Easy;E:p Ad Track-Pdx1-Ngn3-Nkx2.2-Pax4-Ad Easy)were successfully constructed and successfully packaged into adenoviruses.On the 4th day after infected canine ADSCs,RT-q PCR and Western blot detection showed that the different foreign genes in each combination were overexpressed.3. The best induction combination(E:p Ad Track-Pdx1-Ngn3-Nkx2.2-Pax4-Ad Easy)was obtained through cell morphology observation,dithizone staining,glucose-stimulated insulin secretion test,cell immunofluorescence and RT-q PCR analysis on the 30th day after canine ADSCs were infected.Under the stimulation of high glucose(25 mmol/L),the insulin secretion volume and intracellular insulin content of this combination can reach to 100.94±3.70 and 109.08±4.88μIU/10~5cells,respectively.In summary,this study successfully constructed of five different groups multi-gene co-expression adenovirus vectors containing Pdx1,Ngn3,Sox9,Pax4,and Nkx2.2 genes,which were packaged as adenovirus venom infected into canine ADSCs and tested for induced differentiation.It was found that the Pdx1,Ngn3,Nkx2.2 and Pax4 gene combination transfection group had the best induction effect.This study provides an experimental basis for further exploring a more efficient scheme for inducing differentiation of canine ADSCs to IPCs.