| Objective1.To compare the biological characteristics of human umbilical cord derived mesenchymal stem cells(HUC-MSCs)and adipose tissue derived mesenchymal stem cells(ADSCs),and to select functional superior stem cells;2.To study the repairing effect of mesenchymal stem cells on islet cells in type 2diabetes mellitus;3.To explore the repairing mechanism of mesenchymal stem cells on islet cells in type 2 diabetes mellitus.Methods1.Immunophenotype,activity,apoptosis rate,proliferation curve,multi differentiation ability and gene expression of P6 HUC-MSCs and ADSCs were detected and compared statistically;2.Islet cells were isolated from 10 type 2 diabetes mellitus(T2DM)donors and10 non-diabetic(ND)donors,and the differences of islet cells production and morphology between the two groups were compared.HUC-MSCs and T2DM islet cells co cultured group and T2DM islet cells isolated group were established in vitro.After 48 hours of culture,the morphology,viability,glucose stimulation index(GSI)and the proportion of insulin positive cells of the two groups were compared;Five type 2 diabetic mice(db/db)were infused with 1×10~6 HUC-MSCs via the tail vein at the 7th week,and then infused with 1×10~6 HUC-MSCs via the tail vein at the 9th week as the treatment group.Five type 2 diabetic mice(db/db)were fed as the control group.All mice were sacrificed at the 11th week.The random blood glucose,fasting insulin and glucose,OGTT test and the ratio of pancreatic insulin positive cells were compared between the two groups;3.Gene chip and qPCR were used to detect the gene expression difference of T2DM islet cells between the single culture group and the co culture group;HUC-MSCs were co cultured with T2DM islet cells or ND islet cells for 24 hours.The expression of IL-1Ra in HUC-MSCs co cultured group and HUC-MSCs cultured alone group was compared;HUC-MSCs were cultured with 0/2.5/5/10 ng/ml IL-1βfor 12 hours,and the expression of IL-1ra in HUC-MSCs was compared;After neutralizing IL-1Ra in the co culture system of T2DM+MSC,the expression of IL-1βgene and islet function genes(Maf A,Nkx6.1)and GSI of T2DM islet cells were compared with those of T2DM alone culture group and T2DM+MSC co culture group;After knockdown of IL-1Ra related genes in MSCs,the difference of IL-1Ra m RNA expression between knockdown group and control group was compared;T2DM group,KD-MSC+T2DM group and NC-MSC+T2DM group were established,and the differences of GSI of T2DM islet cells among the three groups were compared.Results1.There was no significant difference in immunophenotype,activity and apoptosis rate between the two groups(P>0.05);The HUC-MSCs proliferation ability of 2 groups of Mesenchymal stem cells(MSCs)cultured after 24h was higher than ADSCs(P<0.05);Both HUC-MSCs and ADSCs had osteogenic,adipogenic and chondrogenic differentiation abilities;qPCR was used to detect the m RNA expression of VEGF,HGF,TGF-β,IGF1 and IDO1 in HUC-MSCs and ADSCs.The results showed that the expression of TGF-βand IDO1 in HUC-MSCs was higher than that in ADSCs(P<0.001);2.T2DM donors and ND donors could extract better islet cells from the pancreas,but T2DM donors had lower islet yields(P<0.05);T2DM islet cells and HUC-MSCs co cultured with 24h and 48h showed no difference in islet cells viability between the2 groups compared with the single culture group(P>0.05),but the morphology of T2DM islet cells in co culture group was better;GSI test showed that the glucose stimulation index of T2DM islet cells in co culture group was higher than that in single culture group at 24 h and 48 h(P<0.05);Insulin immunofluorescence staining showed that the proportion of insulin positive cells in co culture group was higher,and the difference was statistically significant(P<0.001).In vivo experiments in mice showed that the results of random glucose,fasting insulin/glucose and OGTT in HUC-MSCs treatment group were significantly better than those in control group(P<0.05);Insulin immunofluorescence staining showed that the percentage of insulin positive cells increased in the treatment group,but the difference was not statistically significant(P>0.05);3.The results of gene chip and qPCR showed that the expression of inflammatory factor related genes were down-regulated and the islet function genes were up-regulated in the MSC+T2DM group.The expression of IL-1ra in HUC-MSCs co cultured with T2DM islets increased significantly(P<0.05);IL-1βcould induce HUC-MSCs to express IL-1ra in a concentration dependent manner(P<0.001).After neutralizing IL-1Ra in co culture system,the expression of IL-1βgene increased(P<0.01),the expression of MAf A/Nk X6.1 decreased(P<0.05),and GSI test showed that the glucose stimulation index also decreased(P<0.01);After knockdown of IL-1Ra gene in HUC-MSCs,the expression of IL-1Ra decreased significantly(P<0.01);Compared with NC-MSC+T2DM group,GSI value of T2DM islet cells decreased significantly in KD-MSC+T2DM group(P<0.001).Conclusions1.Comparing the 6 biological characteristics of HUC-MSCs and ADSCs,including immunophenotype,cell activity,apoptosis rate,proliferation ability,multidifferentiation ability and gene expression,HUC-MSCs has some advantages in proliferation and gene expression;2.Both in vitro co culture and in vivo animal experiments confirmed that HUC-MSCs could improve the function of T2DM islet cells and repair the islet cells;3.Islet cells of T2DM donors can express a variety of inflammatory factors,the inflammatory state of islet cells can induce HUC-MSCs to express IL-1ra,IL-1ra can inhibit the inflammatory response of islet cells,up-regulate the expression of islet function genes,restore the function of islet cells. |
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