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

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) m RNA、骨涎蛋白(BSP)m RNA水平均显著升高,Runt相关基因(Runx)2 m RNA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 m RNA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。

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.