Effects And Mechanism Of Senolytics On The Stemness Maintenance,Osteogenic Differentiation And Replicative Aging Of Human Periodontal Ligament Stem Cells

Background and purpose:This project aims to explore the regulation of Senolytics(Quercetin and Dasatinib)on stemness maintenance,osteogenic differentiation and replicative aging of hPDLSCs,and to select the optimal concentration and ratio of Quercetin(Q)and Dasatinib(D).The possible mechanism of Senolytics regulating the biological characteristics of hPDLSCs was preliminarily discussed.Methods:Primary hPDLSCs cells were obtained by tissue block adherent method.hPDLSCs were cultured in vitro and induced into osteogenesis and lipids.The multidirectional differentiation potential of hPDLSCs was identified by alkaline phosphatase,alizarine red and oil-red O staining.Cloning and proliferation were identified by crystal violet staining.Surface mesenchymal stem cell markers were detected by flow cytometry.Cytotoxic effects of Q and D on cells were detected by CCK8.The optimal concentration of Q and D was screened by ALP activity and qRT-PCR.ALP and alizarin red staining were used to identify the optimal concentration of Senolytics(D+Q).Western Blot that was used to detect osteogenic related proteins(COL 1,ALP,RUNX2),ALP activity,ALP and alizarin red staining were used to verify the effect of Senolytics on osteogenic differentiation.Western Blot was used to detect stemness maintenance related proteins(C-MYC,NANOG,OCT-4),so that we could verify the effect of Senolytics on stemness maintenance of hPDLSCs.The hPDLSCs with good growth status were selected to be amplified into the 20st generation(P20-hPDLSCs)in vitro,and the natural aging model was established.The fifth generation hPDLSCs(P5-hPDLSCs)were taken as control.Senescence associated β-galactosidase(SA-β-Gal)staining and qRT-PCR that was used to detect the expression of senescence related genes P16 and P21 were used to verify the effect of Senolytics on natural aging of hPDLSCs.Western Blot was used to detect the effect of Senolytics on Hippo-YAP/TAZ.The specific knockdown TAZ model was established by lentivirus transfection.The role of TAZ in Senolytics in promoting hPDLSCs osteogenesis was verified by ALP activity detection,ALP staining detection,alizarin red staining detection and Western Blot detection of osteogenic related protein expression.YAP inhibitor Verteporfin has been used to inhibit the expression of YAP in hPDLSCs.Western Blot was used to detect the expression of stemness related proteins and verify the role of YAP in Senolytics in promoting stemness maintenance of hPDLSCs.Results:hPDLSCs that met the requirements of subsequent experiments were successfully isolated,extracted and amplified.Q in a certain concentration range can effectively promote the osteogenic differentiation of hPDLSCs.The best concentration of Q was 2.5 uM.Concentrations ≤1 nM D promoted osteogenesis of hPDLSCs,while concentrations>1 nM inhibited osteogenesis.The optimal concentration of D is 1 nM.The combination of Senolytics enhanced the osteogenic effect of Q and changed the effect of high concentration of dasatinib on osteogenesis.Finally,D(1 nM)+Q(2.5 uM)and D(10 nM)+Q(2.5 uM)were selected for subsequent experiments.Senolytics promoted the expression of osteogenic proteins,ALP activity,ALP staining,and the formation of alizarin red mineralized nodules.Senolytics promotes the expression of related stemness maintenance proteins.Senolytics can effectively down-regulate the expression of senescence related genes and the proportion of β-galactosidase positive cells by eliminating senescent cells.Senolytics up-regulated the protein expression of YAP/TAZ.Knockdown of TAZ partially reversed the osteogenic effect of Senolytics on hPDLSCs.Inhibition of YAP expression partially reversed the positive regulation of Senolytics on stemness maintenance of hPDLSCs.Conclusion:Senolytics(D+Q)can positively regulate osteogenic differentiation and stemness maintenance of hPDLSCs,and can resist replicative senescence of hPDLSCs to a certain extent.Hippo-YAP/TAZ participates in regulating the effects of Senolytics on the biological characteristics of hPDLSCs.