| Diabetes,including type 1 and type 2 diabetes,is the third most serious health-threatening disease in the world.Islet transplantation is an effective strategy for the treatment of diabetes.However,the source of pancreatic islets is limited,and there is an urgent need to produce more insulin producing cells(IPCs).The use of transgenic technology to induce stem cells to differentiate into insulin-secreting cells has broad prospects.Adipose-derived mesenchymal stem cells(AMSCs)can be obtained in large quantities at low risk and have higher immune regulation capabilities,making them an ideal source of seed cells.Studies have shown that the induction efficiency of single gene transfection is not high.Therefore,in this study,we first verified the role of Hnf1b gene in inducing the differentiation of canine AMSCs into IPCs in vitro,and on this basis,combined with the cascade regulators Pdx1,Ngn3,Pax4,Nkx6.1,which play a key role in the development of islet cells,constitute three different multi-gene co-expression adenovirus vectors.Then three multi-gene co-expression adenovirus reprograms canine AMSCs to differentiate into IPCs.The induction efficiency was detected by RT-q PCR,Western-blot,cell morphology observation,dithizone staining,glucose-stimulated insulin secretion test,etc.,and the best induction combination was selected by comparison.The results of the study are as follows:1.The adenovirus expression vector pAdEasy-Hnf1b was constructed,and the Hnf1b si RNA was designed to be transfected into canine AMSCs with inducers.After 25 days,the results showed that both the overexpression group and the silence group formed pancreatic islet-like cell clusters.The overexpression group’s diameter and the number is large,the staining of dithizone is darker;The amount of insulin secreted by the overexpression group under high glucose stimulation is 158.27±9.86μIU/10~5 cells,which is significantly higher than the control group(125.23±4.35μIU/10~5cells)(P<0.001);The expression of pancreatic islet cell development-related genes Nkx6.1 and Ins in the overexpression group was significantly up-regulated compared with the control group(P<0.001);The amount of insulin secreted by the silent group under high glucose stimulation is 102.36±4.36μIU/10~5 cells,which is significantly lower than the control group(P<0.001);The expression of pancreatic islet cell development-related genes Pdx1,Maf A,Nkx6.1,Pcsk2,Ins in the silent group was significantly down-regulated compared with the control group(P<0.001);The cell immunofluorescence results of the overexpression group showed secretion of C-peptide and insulin.2.Three combinations of multi-gene co-expression vectors were constructed,pAdEasy-Hnf1b-Pdx1-Ngn3(A),pAdEasy-Hnf1b-Pdx1-Ngn3-Pax4(B),pAdEasy-Hnf1b-Pdx1-Ngn3-Nkx6.1(C).Four days after transfection of 293A cells,RT-q PCR and Western-blot results showed that the target genes of each group were expressed.The three multi-gene co-expression adenovirus infects canine AMSCs.After 25 days,pancreatic islet-like cell clusters were formed in group A,B,and C.Group B had the largest number,and dithizone staining was the deepest.The amount of insulin secretion in group B under high glucose stimulation was 140.79±3.69μIU/10~5 cells,which was significantly higher than group A(64.84±5.36μIU/10~5cells)and group C(120.22±2.15μIU/10~5cells)(P<0.001);The detection results of the expression of genes related to the development of pancreatic islet cells showed that,except for the Nkx2.2gene,the remaining genes of group A,group B,and group C were significantly higher than those of the empty vector group(P<0.001);The m RNA expression of Pdx1 gene in group B was significantly higher than that of group A(P<0.01)and higher than that of group C(P<0.05);The Pcsk1 gene m RNA expression in group B was significantly higher than that in group C(P<0.001);The m RNA expression of Ins gene in group B was higher than that in group A(P<0.05),and there was no significant difference from group C.In summary,Hnf1b gene can promote the differentiation of canine AMSCs into IPCs in vitro.Three different combinations of Hnf1b,Pdx1,Ngn3,Pax4 and Nkx6.1 were transfected into canine AMSCs to induce differentiation into IPCs.It is shown that the combination of Hnf1b,Pdx1,Ngn3 and Pax4 genes has the best induction efficiency,which provides a new reference for exploring more efficient methods of stem cell differentiation into IPCs,and provides new ideas for clinical treatment of diabetes. |
PDF Full Text Request