张应力刺激下Wnt/β-catenin信号通路对成牙骨质细胞Runx2表达调控的体外研究

目的 研究张应力刺激下Wnt/β-catenin信号通路对成牙骨质细胞Runx2表达的调控。方法 以成牙骨质细胞样细胞OCCM30为研究对象，应用FX4000T应力加载装置对其加载张应力，时间为0、3、6、12 h，然后分别使用不同质量浓度的Dikkopf-1（DKK1）处理48 h，采用Western blot法检测β-catenin蛋白的表达水平，实时荧光定量聚合酶链反应法（RT-PCR）检测Runx2 mRNA的表达水平。选取最佳作用时间点（12 h）和最佳阻断剂质量浓度（150 ng?mL-1），将样本分为A、B、C、D共4组，A组不加力不加阻断剂，B组加力加阻断剂，C组加力不加阻断剂，D组不加力加阻断剂，测量β-catenin蛋白和Runx2 mRNA的表达水平。结果 1）张应力加载3、6、12 h后，Runx2 mRNA表达水平和β-catenin蛋白水平与对照组相比均明显升高，差异有统计学意义（P<0.05）。2）不同质量浓度DKK1处理后，细胞内Runx2 mRNA的表达量呈下降趋势。3）未加载张应力的情况下，DKK1明显抑制了β-catenin蛋白的表达，Wnt/β-catenin通路被抑制；加载张应力情况下，DKK1处理组β-catenin蛋白表达低于未加DKK1处理组；不加阻断剂的情况下，张应力刺激增强了β-catenin蛋白的表达；阻断剂作用后，应力刺激组β-catenin蛋白表达相对较高。结论 机械应力刺激下Wnt/β-catenin信号通路可正向调控成牙骨质细胞Runx2基因的表达。

Investigation of Wnt/β-catenin signaling pathway on regulation of Runx2 in cementoblasts under mechanical stress in vitro

Objective  Periodontal tissue remodeling includes remodeling of alveolar bone, periodontal ligament, and cementum. Cementoblast plays a main role in repairing root resorption. Canonical Wnt/β-catenin signaling can promote the odontogenic differentiation in osteoblast. However, the mechanism on how the orthodontic force influences the function of cementoblast and the relationship between the canonical Wnt/β-catenin signaling and Runx2 of cementoblast are not yet known. The aim of this study is focus on this relationship. Methods  OCCM30 cementoblasts were subjected to mechanical strain by four-point bending system with tension stress for 0, 3, 6, and 12 h. They were pretreated with different concentrations of Dikkopf-1 (DKK1) for 48 h. Western blot analysis was performed to detect the β-catenin levels in the nucleus. Runx2 mRNA was observed by real-time quantitative polymerase chain reaction (RT-PCR). OCCM30 cementoblasts were then pretreated with 150 ng?mL-1 DKK1 for 48 h and subjected to mechanical strain by FX4000T system with tension stress for 12 h. Western blot analysis was conducted to detect the β-catenin levels in the nucleus, and Runx2 mRNA was observed by RT-PCR. Results  OCCM30 cementoblasts had significantly higher Runx2 mRNA and β-catenin levels after being loaded with mechanical stress. The amount of Runx2 mRNA in OCCM30 cementoblasts was significantly decreased by DKK1. When OCCM30 cementoblasts were pretreated with DKK1 without stress, their β-catenin level was significantly decreased by DKK1 and Wnt signaling was blocked. When they were not pretreated with stress, the β-catenin level with DKK1 was lower than that without DKK1.Without DKK1, the β-catenin level in OCCM30 cementoblasts increased after being loaded with mechanical stress. With DKK1, the β-catenin level in OCCM30 cementoblasts, which were loaded with mechanical stress, was higher than that without mechanical stress. Conclusion  Cementoblasts had higher Runx2 mRNA expression under mechanical stress because of the Wnt/β-catenin signaling pathway effect.