The Role And Mechanism Of SERPINB2 Gene Regulating Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

This study is divided into three parts:(1)Study on endogenous expression of SERPINB2 in bone marrow mesenchymal stem cells;(2)The effect of SERPINB2 on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and the possibility of its application in fracture treatment;(3)The specific mechanism of SERPINB2 on osteogenic differentiation of BMSCs.PurposeWith the development of industry and increase of human aging-trend,the number of patients with fracture increases,and the bone defect caused by fracture as well as fracture has become an important clinical problem.A potential strategy to promote fracture healing is to transplant or regulate BMSCs.It is very important to understand the process of BMSCs osteogenic differentiation and the regulation of related signaling pathways for its clinical application.The proteins of Serine Protease Inhibitor(Serpin)family plays an important role in physiological processes that require a high degree of regulation in organisms,such as coagulation,fibrinolysis,stress and inflammation,and is also associated with bone formation.As a member of the Serpins,SERPINB2 has two forms,a secretory protein and another intracellular protein.And SERPINB2 is highly expressed in monocytes and macrophages,mainly upregulated in response to injury and stress.The specific function of SERPINB2 is still unclear.Its expression in BMSCs and its role in osteogenic differentiation are still unclear.Therefore,this study aims to explore the function and mechanism of SERPINB on osteogenic differentiation of BMSCs.MethodsGene chip GSE152677 was retrieved and downloaded from GEO database.And the expression of SERPINB2 gene after fracture was obtained by analyzing the data with RStudio software.In addition,human bone marrow mesenchymal stem cells were extracted,cultured and identified to verify the endogenous expression of SERPINB2 gene in the process of osteogenic differentiation,and to preliminarily explore the effect of SERPINB2 on human bone marrow mesenchymal stem cells and the possibility of SERPINB2 as a therapeutic target for fracture healing.The effect of Serpin B2 gene on the proliferation and osteogenic differentiation of human BMSCs was investigated by two methods of exogenous human Serpin B2 recombinant protein and small interfering RNA inhibition of Serpin B2 gene expression.The proliferation was detected by CCK-8 assay,and the osteogenesis was tested by RT-PCR,Western Blot,immunofluorescence,alkaline phosphatase staining,and Aizarin red staining.Then,the effect of inhibiting SERPINB2 gene expression on fracture healing in a mouse model of tibia fracture was verified.Finally,the signal pathway expression was detected by Western Blot.The Wnt/β-catenin pathway inhibitor DKK-1 was added to verify the effect of SERPINB2 on bone marrow mesenchymal stem cell osteogenic differentiation through the Wnt pathway.ResultsOur first study found that SERPINB2 is endogenously expressed in BMSCs,and its expression gradually decreases during osteogenic differentiation.Subsequently,we found that exogenous recombinant SERPINB2 protein inhibited the expression of osteogenic genes and proteins.Inhibition of SERPINB2 gene expression by transfection with small interfering RNA significantly promoted the expression of osteogenic related genes and proteins,such as Runx2,SP7,and COL1A1.And it also increased the formation of alkaline phosphatase,a marker of early osteogenesis,and calcium nodules,a marker of late osteogenesis.In the model of mice tibial fracture,we found that covering the fracture with small interfering RNA of SERPINB2 combined with a atelocollagen gel significantly promoted bone formation.Subsequently,we investigated the mechanism of SERPINB2 gene affecting osteogenic differentiation of human BMSCs,and found that inhibition of SERPINB2 gene can activate the Wnt/β-catenin pathway of human BMSCs.Adding the inhibitor of Wnt pathway DKK-1 significantly reduced the enhancing effect of SERPINB2 gene expression on osteoblastic differentiation of human BMSCs.ConclusionInhibition of SERPINB2 gene expression promoted osteogenic differentiation of BMSCs by activating Wnt/β-catenin.Inhibition of SERPINB2 in combination with the terminating peptide collagen gel promotes fracture healing.