细胞外信号调节蛋白激酶1/2在介导周期性牵张力对牙周膜细胞成骨分化中的作用
The role of extracellular signal regulated kinase 1/2 in mediating osteodifferentiation of human periodontal ligament cells induced by cyclic stretch
作者：宋京, 任大鹏, 颜世果, 蓝菁, 袁晓, 郭庆圆, 戚向敏
Author：Jing Song, Dapeng Ren, Shiguo Yan, Jing Lan, Xiao Yuan, Qingyuan Guo, Xiang-min Qi
收稿日期：2016-08-11 年卷（期）页码：2017,35(5):520-520-526
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：牙周膜细胞,成骨分化,周期性牵张力,细胞外信号调节蛋白激酶1/2,
Key words：periodontal ligament cells,osteogenic differentiation,cyclic stretch,extracellular signal regulated kinase 1/2,
基金项目：国家自然科学基金（31170891）;山东省卫生厅重点基金（2011HD001）
中文摘要
目的 研究周期性牵张力刺激下细胞外信号调节蛋白激酶（ERK）1/2对牙周膜细胞成骨分化的分子调控机制。方法 组织块法培养人牙周膜细胞。采用多通道应力加载系统对细胞施加频率0.5 Hz、振幅10%的周期性牵张力（加力时间1、3、6、12、24 h）,以不加力的细胞作为对照,并分别在加力前应用ERK1/2通路特异性抑制剂U0126以及对细胞转染ERK1/2显性负相变异体（DN-ERK1/2）。采用实时荧光定量聚合酶链式反应（real-time PCR）及蛋白质印迹法研究人牙周膜细胞的基因蛋白水平变化。结果 加力后人牙周膜细胞的p-ERK1/2蛋白水平及骨钙蛋白（OCN） mRNA、骨涎蛋白（BSP）mRNA水平均显著升高,Runt相关基因（Runx）2 mRNA及蛋白水平在加力3、6 h均显著升高。加入抑制剂U0126或细胞转染DN-ERK1/2后,Runx2、OCN、BSP mRNA水平以及Runx2、p-ERK1/2蛋白水平均降低。结论 ERK1/2是周期性牵张力刺激下牙周膜细胞成骨分化的重要分子途径,力学刺激下激活的ERK1/2可能通过提高Runx2蛋白的表达水平而参与成骨基因OCN和BSP的转录表达。
英文摘要
ObjectiveThis study aimed to investigate the mechanism of cyclic stretch that promotesthe osteogenic diffe-rentiation of human periodontal ligament cells (hPDLCs) through the mediation of extracellular-signal-regulated kinase 1/2 (ERK1/2).MethodshPDLCs were isolated throughthe explant method and culturedin vitro. hPDLCs were mechanically stimulated by a multi-channel cell-stress-loading system for 1, 3, 6, 12, and 24 h. The magnitude of stretch was 10% defor-mation, and the frequency was 0.5 Hz. Nonloaded cells were used as control group. ERK1/2 activation was blocked by U0126, a specific ERK1/2 pathway inhibitor. Additionally, hPDLCs were transfected with adenoviral vector encoding dominant negative ERK1/2 (DN-ERK1/2) to continuouslyinhibit ERK1/2 activation. The mRNA and protein levels of target geneswere detected through real-time polymerase chain reaction and Western blot.ResultsCyclic stretching promoted the expression of ERK1/2, osteocalcin (OCN) mRNA, and bone sialoprotein (BSP) mRNA. The expression of runt-related transcription factor (Runx) 2 protein and mRNA also increased at 3 and 6 h of cyclic stretching. The inhibition of ERK1/2 by U0126 and DN-ERK1/2 suppressed the expressionof Runx2 mRNA, OCN mRNA, BSP mRNA, Runx 2 protein, and p-ERK1/2 protein relative to that in stretched cells without the ERK1/2 inhibitor.ConclusionERK1/2 is a critical molecule in the mediation ofthe osteogenic differentiation of hPDLCs under mechanical stimulation. ERK1/2 activation induced the elevation of Runx2 protein levels, which may be involved in the stretch-induced expressions of OCN and BSP.
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