Effects Of IGFBP5 And KDM6A On Osteogenic Differentiation Of PDLSCs Under Stimulation Of Inflammatory Cytokines

Objective: To study the influence and molecular mechanism of insulin-like growth factor-binding protein 5(IGFBP5)and lysine(K)-specific demethylase 6A(KDM6A)on osteogenic differentiation of periodontal ligament stem cells(PDLSCs)under inflammatory microenvironment,to provide a new therapeutic target for periodontal tissue regeneration in inflammatory microenvironment.Method: 1.After PDLSCs were collected and isolated,their surface antigen markers were detected by flow cytometry.2.After the stable IGFBP5 knock-down cell line and KDM6 A knock-down cell line were established,the cells were cultured in osteogenic differentiation medium.With the stimulation of tumor necrosis factor α(TNF-α)and interleukin(IL)-17,the viability was detected by using cell counting kit-8(CCK-8),the apoptosis was analyzed by Annexin V,and differentiation and biological characters were detected by alkaline phosphatase(ALP)activity,ALP staining and alizarin red staining.3.After osteogenic differentiation of IGFBP5 knock-down PDLSCs in vitro,we use real time polymerase chain reaction(PCR)to detect the influence of inflammatory cytokines on m RNA expression of marker genes relating to osteogenic differentiation and histone methyltransferase.We also detect the m RNA expression of marker genes relating to osteogenic differentiation of the PDLSCs with KDM6 A knock-down in vitro and use flow cytometry microsphere capture chip technology to analysis the change of related inflammation factors.4.After osteogenic differentiation of KDM6 A knock-down PDLSCs in vitro,we utilize Western Blot to detect the influence of inflammatory cytokines on protein expression of p-p65、β-catenin.Result: 1.After IGFBP5 knock-down cell line cultured in osteogenic differentiation medium was stimulated by cytokines,the ALP staining weakened.ALP activity was lower after stimulated by TNF-α and IL-17.The results of real time PCR revealed that the expression of marker genes osteocalcin(OCN)and bone sialoprotein(BSP)significantly decreased in osteogenic differentiation group stimulated by TNF-α and IL-17.Also,the level of lysine(K)-specific demethylase 2A(KDM2A)、lysine(K)-specific demethylase 5B(KDM5B)、 lysine(K)-specific demethylase 3B(KDM3B)and Arginine demethylase and lysine hydroxylase 6(JMJD6)shared the similar trend.(P<0.05,n=3)2.ALP staining showed that the staining was lighter in PDLSCs with knockdown of KDM6 A after adding TNF-α and IL-17 stimulants.Alizarin red staining showed that no significant change in staining.The results of cell proliferation assay showed that there was no significant difference between the control group and osteogenic differentiation group stimulated by TNF-α、IL-17.The results of cell flow cytometry demonstrated that the cell apoptosis rate slight increased after KDM6 A knockdown.Adding TNF-α and IL-17 enhanced the cell apoptosis rate.PCR results showed that TNF-α and IL-17 significantly inhibited the m RNA expression of RUNX2,BSP and OCN compared with those in the control group.Flow cytometry showed that inflammatory factor IL-6 significantly increased after knock-down of KDM6 A in PDLSCs.(P<0.05,n=3)3.Western Blot results illustrated that TNF-α and IL-17 decreased β-catenin level in cytoplasm of KDM6 A knockdown PDLSCs.Conclusion: 1.It was verified that IGFBP5 could induced the osteogenic differentiation of PDLSCs with inflammatory factors TNF-α and IL-17 stimulated.2.It was verified that that KDM6 A could induced the osteogenic differentiation of PDLSCs with inflammatory factors TNF-α and IL-17 stimulated.3.It was verified that that KDM6 A could induced the osteogenic differentiation of PDLSCs with inflammatory factors TNF-α and IL-17 stimulated may be related to the activation of Wnt/β-catenin signaling pathway.