牵张应力刺激下Erk1/2在人牙周膜细胞成骨化的作用分析

Effect of Erk1/2 on osteogenesis of human periodontal ligament cells stimulated by distraction stress

研究牵张应力刺激下细胞外调节蛋白激酶(Erk)1/2在人牙周膜细胞成骨化的作用,本实验收集行口腔正畸治疗拔除的健康前磨牙,组织块法和消化法原代培养人牙周膜细胞。振幅10%、频率0.5Hz的周期性牵张应力刺激细胞,以不加牵张应力的细胞为对照组;并于牵张应力刺激前分别使用ERK1/2通路抑制剂PD98059处理细胞,ERK1/2显性负性突变体转染细胞。实验结果显示,牵张应力刺激不同时间p-ERK1/2蛋白表达水平均明显提升(P<0.05),其中以刺激1 h、3 h时p-ERK1/2蛋白表达水平最高;ERK1/2蛋白表达水平在牵张应力刺激不同时间差异无统计学意义(P>0.05);牵张应力刺激3、6 h时Runt相关基因2蛋白表达水平明显提升(P<0.05)。加入抑制剂PD98059或ERK1/2显性负性突变体转染后,Runt相关基因2、碱性磷酸酶(ALP)、成骨相关基因骨钙素(OCN mRNA)表达水平明显下调;p-ERK1/2、Runt相关基因2蛋白表达水平明显下调。实验证明,Erk1/2信号通路在牵张应力刺激下牙周膜细胞成功分化中发挥重要作用,Erk1/2被力学刺激激活后可通过上调Runt相关基因2蛋白表达介导成骨相关基因ALP、OCN的表达。

To investigate the effect of extracellular regulated protein kinase(Erk) 1/2 on osteogenesis of human periodontal ligament cells stimulated by stretch stress. Human periodontal ligament cells were cultured from healthy premolars extracted by orthodontic treatment in our hospital. Cells were stimulated by cyclic stretch stress with amplitude of 10% and frequency of 0.5 Hz, and then treated with PD98059, an ERK1/2 pathway inhibitor, before stretch stress stimulation. ERK1/2 dominant negative mutant was transfected into cells. The Results showed that the expression level of p-ERK1/2 protein increased significantly at different time of stretch stress stimulation(P<0.05), and the expression level of p-ERK1/2 protein was the highest at 1 h and 3 h of stretch stress stimulation;the expression level of ERK1/2 protein had no significant difference at different time of stretch stress stimulation(P>0.05);the expression level of Runt-related gene 2 protein increased significantly at 3 h and 6 h of stretch stress stimulation(P<0.05). After transfection with PD98059 or ERK1/2 dominant negative mutant, the expression levels of Runt-related genes 2, ALP, OCN and p-ERK1/2 and Runt-related genes 2 were significantly down-regulated.So Erk1/2 signaling pathway plays an important role in the successful differentiation of periodontal ligament cells stimulated by stretch stress. Erk1/2 can be activated by mechanical stimulation, which can mediate the expression of ALP and OCN by up-regulating the expression of Runt-related gene 2.