| Diabetes is a chronic metabolic disease ubiquitous all over the world;it has a severe impact on people’s health and medical resources.At present,islet transplantation is a promising treatment for diabetes,but it is limited due to the shortage of islet donors and immune rejection problems.Therefore,finding other sources of Insulin producing cells(IPCs)for transplantation has become an effective way to treat diabetes.In recent years,although initial results have been proved in inducing stem cells to differentiate into IPCs in vitro,the degree of differentiation of IPCs and the measure of Insulin secretion were very low.Through searching the literature,we found that Pdx1,Ngn3,Pax4 and Nkx2.2 all played a certain role in the development and maturation of pancreatic islets.Studies have also shown that the transfer of these genes into stem cells alone can promote their differentiation into IPCs,but the differentiation efficiency was not stable enough,and the Insulin secretion of IPCs was low and did not change with changes in glucose concentration.Our laboratory previously screened out the gene Dll1,which may play a role in inducing the differentiation of canine adipose MSCs to IPCs through sequencing analysis.We started with the study of the role of Dll1 gene in inducing the differentiation of canine adipose MSCs to IPCs for further studying Dll1 and its connection with Pdx1,Ngn3,Pax4 or Nkx2.2 to form a poly-combined transfection to screen the effects of different combinations reprogramming canine adipose MSCs into IPCs.The main results of the study are as follows:1.In order to study the role of Dll1 gene in inducing the differentiation of canine adipose MSCs into IPCs,we overexpressed Dll1 in canine adipose MSCs and silenced the Dll1 gene by si RNA,plus an induction program to induce canine adipose MSCs to differentiate into IPCs.Dll1 overexpression group results:(1)RT-q PCR detection showed that the expression of Maf A,Nkx6.1 and Ins genes in the overexpression Dll1 group was very significantly higher(P<0.001)than in the control group(48.975±3.578 vs 33.975±3.453;55.156±6.267 vs 25.106±1.267;39.258±3.752 vs 21.533±2.401);Pdx1,Maf A,Pcsk2 and Ins gene expression in the Dll1 silence group were very significantly lower(P<0.001)than the control group(4.388±0.898 vs 15.997±1.660;9.365±1.356 vs 33.975±3.453;16.358±1.258 vs 27.538±2.935;12.397±0.369 vs 21.533±2.401).(2)Dithizone staining,the cell clusters in the overexpression group were scarlet.(3)Immunofluorescence detection showed that the cell clusters in the overexpression group secreted Insulin.(4)High glucose stimμLation,the Insulin secretion in the Dll1 overexpression group(160.25±10.35μIU/10~5 cells)was significantly higher(P<0.001)than the Insulin secretion in the control group(125.23±4.35μIU/10~5 cells),the amount of Insulin secretion in the Dll1 silent group(101.367±5.367μIU/10~5 cells)was very significantly lower(P<0.001)than that in the control group;The Insulin secretion of each combination under KCl stimμLation was similar to that under high glucose stimμLation.The Insulin stimμLation release index SI(2.70±0.05)of the overexpression Dll1 group was significantly higher than(P<0.01)the control group SI(2.36±0.11),and the Insulin stimμLation release index SI(2.16±0.08)after Dll1 silence was very significantly lower than the control(P<0.001)Group SI.2.Construct three sets of adenovirus mμLti-gene co-expression shuttle vectors p Ad Track-CMV-Dll1-Pdx1-Ngn3,p Ad Track-CMV-Dll1-Pdx1-Ngn3-Pax4 and p Ad Track-CMV-Dll1-Pdx1-Ngn3-Nkx2.2,after verification were correct,and they were recombined with p Ad Easy-1 to obtain recombinant adenovirus mμLti-gene co-expression vector p Ad Easy-Dll1-Pdx1-Ngn3(combination one),p Ad Easy-Dll1-Pdx1-Ngn3-Pax4(Combination two)and p Ad Easy-Dll1-Pdx1-Ngn3-Nkx2.2(combination three),and they were verified to infect 293A cells.(1)RT-qPCR showed the expression levels of Maf A,Nkx6.1 and Ins in the overexpressed Dll1 group were significantly higher than those in the control group(48.975±3.578 vs 33.975±3.453;55.156±6.267 vs 25.106±1.267;39.258±3.752 vs 21.533±2.401);The gene expression levels of Pdx1,Maf A,Pcsk2 and Ins in Dll1 silencing group were significantly lower than those in control group(4.388±0.898 vs 15.997±1.660;9.365±1.356 vs 33.975±3.453;16.358±1.258 vs 27.538±2.935;12.397±0.369 vs 21.533±2.401).(2)The Western-blots of the three groups all expressed the protein of the target genes.3.The three groups of recombinant adenovirus venom were respectively infected with canine adipose MSC,and the induction program was combined to transfect the reprogrammed canine adipose MSCs to become IPCs.(1)The three combinations all expressed GFP after 48 hours of infection.After 25 days of cμLture,the number of cell clusters in combination two was the largest.There were about121±6 cell clusters in every 10~6 canine adipose MSCs,and the diameter of the cell cluster was about 100±9μm.There are about 115±14 cells in each cell cluster(n=3).(2)Dithizone staining,the cell clusters in the three combinations are all scarlet,but the second combination is the darkest.(3)RT-q PCR detection,combination two Pdx1,Maf A,Nkx6.1,Pax4,Pcsk1,Pcsk2,Ins gene expression levels were higher than the other two groups,combination two Pdx1,Maf A gene expression levels were significantly higher(P<0.01)than combination one(29.785±2.155 vs 21.095±2.125;24.578±2.135 vs 15.888±1.105),the expression levels of Pdx1,Maf A,Pax4,Pcsk1,and Ins genes in combination two were very significantly lower than(P<0.001)that of islet cells.(4)High glucose stimμLation,the Insulin secretion of combination one(58.51±3.64μIU/10~5cells),combination two(135.97±4.26μIU/10~5cells),and combination three(113±3.26μIU/10~5cells),the secretion of combination two was significantly higher(P<0.01)than that of combination one and combination two,but the secretion of Insulin under high glucose stimμLation was very significantly lower(P<0.001)than that of pancreatic islet cells.The Insulin-stimμLated release index SI(2.33±0.09)of combination two was also higher than the other two groups,but significantly lower(P<0.01)than the pancreatic islet cell SI(2.71±0.08).The results showed that Dll1 gene expression coμLd promote the differentiation of canine adipose MSCs into IPCs in vitro.All three combinations coμLd reprogram MSCs into IPCs,and the reprogramming effect of the four-gene combinations were far better than that of the three-gene combination,among the three-gene combinations,the reprogramming effect of combination two was better than combination three. |
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