华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (5): 520-526.doi: 10.7518/hxkq.2017.05.015

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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

宋京1(), 任大鹏2, 颜世果1, 蓝菁1, 袁晓2,3, 郭庆圆3, 戚向敏1()   

  1. 1.山东大学口腔医院,山东省口腔生物医学重点实验室,济南 250012
    2.青岛大学口腔医学院,青岛大学医学院附属医院正畸科,青岛 266003
    3.青岛市立医院口腔医学中心正畸科,青岛 266075
  • 收稿日期:2016-08-11 修回日期:2016-12-09 出版日期:2017-10-01 发布日期:2017-10-01
  • 作者简介:

    宋京,住院医师,硕士,E-mail:jinglydentist@163.com

  • 基金资助:
    国家自然科学基金(31170891);山东省卫生厅重点基金(2011HD001)

The role of extracellular signal regulated kinase 1/2 in mediating osteodifferentiation of human periodontal ligament cells induced by cyclic stretch

Jing Song1(), Dapeng Ren2, Shiguo Yan1, Jing Lan1, Xiao Yuan2,3, Qingyuan Guo3, Xiang-min Qi1()   

  1. 1. Stomatology Hospital of Shandong University, Shandong Provincial Key Laboratory of Oral Biomedicine, Jinan 250012, China
    2. Stomatology College of Qingdao University; Dept. of Orthodontics, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China
    3. Dept. of Orthodontics, Stomatological Center, The Affiliated Qingdao Municipal Hospital, Qingdao 266075, China
  • Received:2016-08-11 Revised:2016-12-09 Online:2017-10-01 Published:2017-10-01
  • Supported by:
    National Natural Science Foundation of China (31170891);Key Fund of Shandong Provincial Health Bureau (2011HD001)

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。

关键词: 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

Abstract:

Objective This 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). Methods hPDLCs were isolated throughthe explant method and cultured in 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. Results Cyclic 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. Conclusion ERK1/2 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.

Key words: periodontal ligament cells, osteogenic differentiation, cyclic stretch, extracellular signal regulated kinase 1/2

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