Overexpression of collagen triple helix repeat-containing protein 1 promotes proliferation and osteogenic differentiation of human periodontal ligament stem cells

Zhou Rui; Zhang Xuesong; Yang Donghong; Jin Yinan; Yang Yuqi; Ye Zhihui

doi:10.12307/2026.693

Abstract：BACKGROUND:Collagen triple helix repeat-containing protein 1 is a positive regulator of bone formation.However,its role and underlying mechanisms in periodontal ligament stem cells remain unclear. OBJECTIVE:To investigate the effects of collagen triple helix repeat-containing protein 1 on the proliferation and osteogenic differentiation of periodontal ligament stem cells and its mechanism of action. METHODS:Human periodontal ligament stem cells were isolated and cultured in vitro,and the cells were transfected with a overexpressed collagen triple helix repeat-containing protein 1 lentiviral vector.The effect of overexpression of collagen triple helix repeat-containing protein 1 on the proliferation activity of periodontal ligament stem cells was determined by CCK-8 assay and flow cytometry.The effect of overexpression of collagen triple helix repeat-containing protein 1 on the osteogenic differentiation of periodontal ligament stem cells was determined by alkaline phosphatase activity and Alizarin red staining.Western blot assay was used to determine the expression of extracellular regulated protein kinase 1/2 and phosphorylated extracellular regulated protein kinase 1/2 after overexpression of collagen triple helix repeat-containing protein 1.Western blot assay and qRT-PCR were used to determine the effect of overexpression of collagen triple helix repeat-containing protein 1 on the expression of osteogenic differentiation-related factors Runt-related transcription factor 2,osteocalcin,and Osterix in periodontal ligament stem cells after blocking the extracellular regulated protein kinase 1/2 signaling pathway. RESULTS AND CONCLUSION:(1)CCK-8 assay and flow cytometry results showed that overexpression of collagen triple helix repeat-containing protein 1 promoted the proliferation of periodontal ligament stem cells.(2)Alkaline phosphatase activity and Alizarin red staining showed that overexpression of collagen triple helix repeat-containing protein 1 promoted the osteogenic differentiation of periodontal ligament stem cells.(3)Western blot assay results showed that overexpression of collagen triple helix repeat-containing protein 1 activated the extracellular regulated protein kinase 1/2 signaling pathway.(4)Western blot assay and qRT-PCR results showed that overexpression of collagen triple helix repeat-containing protein 1 promoted the expression of Runt-related transcription factor 2,osteocalcin,and Osterix protein and mRNA.When the extracellular regulated protein kinase signaling pathway-specific inhibitor PD98059 was used to inhibit signaling pathway activation,the upregulation of osteogenic-related factors was suppressed to a certain extent.These results suggest that overexpression of collagen triple helix repeat-containing protein 1 can promote the proliferation and osteogenic differentiation of periodontal ligament stem cells,and its osteogenic differentiation-promoting effect may be related to the activation of the extracellular regulated protein kinase 1/2 signaling pathway.