背景:骨凝集素是在骨组织中表达的C型凝集素结构域超家族的分泌型糖蛋白,据报道是一种促进骨再生的新型成骨因子。然而,骨凝素对人牙髓细胞(hDPCs)的影响尚未见报道。因此,我们旨在研究骨凝素在hDPCs中的成牙本质分化,并进一步试图揭示其潜在的机制。
方法:采用细胞毒性试验检测骨凝素的细胞毒性。通过碱性磷酸酶活性测量hDPCs的成牙本质分化及其潜在机制,矿化斑点的形成,碱性磷酸酶(ALP)染色检测牙本质细胞分化的基因和蛋白表达,茜素红S染色,定量实时聚合酶链反应(qPCR),和蛋白质印迹分析,分别。
结果:WST-1分析显示,浓度低于300ng/ml的骨凝集素对hDPC无细胞毒性且安全。以下实验证明骨凝素可以增加ALP活性,加速矿化过程,并在基因和蛋白质水平上上调牙源性分化标志物(P<0.05)。骨粘连蛋白刺激ERK的磷酸化,JNK,和AKT在hDPC中。ERK,JNK,和AKT抑制剂降低了ALP活性和矿化能力,并抑制了牙本质唾液酸磷蛋白(DSPP)和牙本质基质蛋白-1(DMP-1)的表达。
结论:骨凝素可促进hDPCs的成牙本质分化,整个过程可能会刺激ERK,JNK,和AKT信号通路通过增加p-ERK,p-JNK,和p-AKT信号。
BACKGROUND: Osteolectin is a secreted glycoprotein of the C-type lectin domain superfamily, expressed in bone tissues and is reported as a novel osteogenic factor that promotes bone regeneration. However, the effect of osteolectin on human dental pulp cells (hDPCs) has not been reported. Therefore, we aimed to investigate the odontoblastic differentiation of osteolectin in hDPCs and further attempt to reveal its underlying mechanism.
METHODS: Cytotoxicity assays were used to detect the cytotoxicity of osteolectin. The odontoblastic differentiation of hDPCs and its underlying mechanisms were measured by the alkaline phosphatase (ALP) activity, mineralized spots formation, and the gene and protein expression of odontoblastic differentiation through ALP staining, Alizarin red S staining, quantitative real-time polymerase chain reaction, and Western blot analysis, respectively.
RESULTS: WST-1 assay showed osteolectin at concentrations below 300 ng/ml was noncytotoxic and safe for hDPCs. The following experiment demonstrated that osteolectin could increase ALP activity, accelerate the mineralization process, and up-regulate the odontogenic differentiation markers in both gene and protein levels (P < .05). Osteolectin stimulated the phosphorylation of ERK, JNK, and Protein kinase B (AKT) in hDPCs. Extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and AKT inhibitors decreased ALP activity and mineralization capacity and suppressed the expression of dentin sialophosphoprotein and dentin matrix protein-1.
CONCLUSIONS: Osteolectin can promote odontoblastic differentiation of hDPCs, and the whole process may stimulate ERK, JNK, and AKT signaling pathways by increasing p-ERK, p-JNK, and p-AKT signals.