Mechanism Of The Contribution Of Sirt1 In The Osteogenic Differentiation Of Dental Pulp Stem Cells

[Objective]To study the effect of Sirt1-Stat3 pathway in osteogenic differentiation process of DPSCs.And to explore the function of Sirt1 in the efficiency of DPSCs' osteogenic differentiation.To provide a theory basis for improving the efficiency of the clinical application of DPSCs in tissue engineering.[Methods]Isolated dental pulp stem cells from healthy dental pulp tissues by both the explant and the enzymatic digestion method.Purify the stem cells with limited dilution.And identified the stem cells using flow-cytometers and multipotent differentiation experiments.Used mineralized solution to induce osteogenic differentiation in the 4th generation of DPSCs in good condition.Detected the expression level of Sirt1 and p-Stat3 by Western blot assay and made quantitative analysis.Added Sirt1 activator and inhibitor to improve and reduce sirtl activity.Then detected the expression level of Sirt1 and p-Stat3 by Western blot assay and made quantitative analysis.Used mineralized solution to induce osteogenic differentiation in the 4th generation of DPSCs in good condition.Added Sirt1 activator and inhibitor to improve and reduce sirtl activity.Added p-Stat3 inhibitor to reduce the phosphorylation of Stat3.Extracted the total protein of the cells in 7th days and 21st days respectively.The expression levels of Sirt1,p-Stat3 and two kinds of osteogenic markers-osteopontin and bone sialoprotein were detected by Western blot.And made quantitative analysis of the bands.Synchronously,processed ALP staining and von Kossa staining in 7th day and 21st day respectively.[Results]We have successfully get the human dental pulp stem cells with self-renewal and multi-directional differentiation ability.And the cells express the specific surface antigen markers.Osteogenesis induction improved Sirt1 expression level,and reduced the phosphorylation of Stat3 in dental pulp cells.P-Stat3 was decreased when adding sirtl activator in the osteogenic differentiation process.On the contrary,p-Stat3 was increased when adding sirtl inhibitor.Compared with the medium group,all of the other group had a higher expression of Sirt1,osteopontin and bone sialoprotein,a lower expression of p-Stat3,a deeper dyeing of ALP and an increase of mineralization nodes.But compared with the mineralized solution group,the expression of phosphorylated Stat3 decreased but the expression of osteopontin and bone sialoprotein increased with the activating of Sirt1,with the ALP dyeing deepened and the mineralization nodes increased.On the contrary,with the inhibition of Sirt1 activity,the expression of phosphorylated Stat3 increased and the expression of osteopontin and bone sialoprotein reduced with the ALP dyeing lightened and the mineralization nodes reduced.[Conclusions]1.We can isolate hDPSCs with good ability to proliferate and differentiate by both the explant and the enzymatic digestion method.2.Osteogenic differentiation increase the expression of Sirt1 and inhibit the expression of p-Stat3.3.In the dental pulp tissues,sirtl regulate Stat3 by inhibiting the phosphorylation of Stat3,and then hold the function of Stat3.4.Sirt1 can promote osteogenic differentiation of dental pulp stem cells by inhibiting the phosphorylation of Stat3.