周期性张应变对人牙周膜细胞迁移的影响

目的 探讨周期性张应变对人牙周膜细胞(human periodontal ligament cells,hPDLC)迁移的影响及其相关机制,为进一步了解机械力对hPDLC功能的影响提供资料.方法 应用FX-4000T细胞应变加载系统,对体外培养的hPDLC施加周期性张应变,牵张幅度分别为10%和20%,加载时间为6 h和24 h,频率均为0.1 Hz,以未加载的静态细胞作为对照组,应用划痕法观察hPDLC的迁移,蛋白质印迹法检测hPDLC的基质金属蛋白酶9(matrix metalloproteinases-9,MMP-9)的表达变化;用细胞外信号调节蛋白激酶(extracellular signal-regulated kinase,ERK)的特异性抑制剂PD98059预处理hPDLC后,在0.1 Hz、10%幅度条件下,加载6 h,检测细胞p-ERK1/2和MMP-9表达的变化.用细胞迁移划痕法观察加载10%和20%幅度张应变,观察24 h后PD98059和MMP家族的抑制剂强力霉素(doxycycline,DOX)对hPDLC迁移的影响.结果 细胞迁移划痕实验结果显示,牵张幅度和加载时间均可显著促进细胞迁移.与静态组相比,施加牵张幅度分别为10%和20%的张应变6 h后,hPDLC迁移变化不明显,但加载24 h后,10%和20%幅度的张应变均显著促进了hPDLC迁移(P<0.05),细胞迁移率分别为(45 ±8)%和(66±14)%,且20%比10%幅度牵张对细胞迁移的促进作用更显著(P<0.05).蛋白印迹法结果显示,周期性张应变促进了hPDLC的MMP-9表达.牵张幅度对细胞MMP-9的表达有显著影响(P<0.05),但加载时间对细胞MMP-9的表达无显著影响.ERK1/2的抑制剂PD98059不仅可以明显抑制张应变诱导的p-ERK1/2活化,而且可通过ERK信号通路明显抑制张应变诱导的细胞MMP-9表达.细胞迁移划痕实验还证实,加载幅度分别10%和20%的张应变24 h后,抑制剂PD98059和DOX均能有效抑制周期性张应变诱导的hPDLC迁移.结论 周期性张应变可通过激活hPDLC的ERK信号通路,促进细胞的MMP-9表达,从而诱导体外培养的hPDLC迁移.

Cyclic strain promotes migration of human periodontal ligament cell via extracellular signal-regulated kinase(ERK) signaling pathway

Objective To study the effect of cyclic strain on migration of human periodontal ligament cell(hPDLC) and underlying mechanism. Methods The cultured hPDLC were subjected to 10% or 20%-elongation magnitude cyclic strain at frequency of 0. 1 Hz by FX-4000T system for 6 or 24 hours-duration respectively, while the static group serves as control, hPDLC migration was assayed by wound healing method. The expressions of matrix metalloproteinases-9 (MMP-9) and p-ERK1/2 in hPDLC without or with cyclic srain were analyzed by Western blotting. To investigate the effect of ERK signaling pathway and MMP-9 on migration of hPDLC, the cells were incubated with PD98059, a specific extracellular signal-regulated kinase (ERK) kinase inhibitor, or doxycycline, a MMP inhibitor. Then the expressions of p-ERK1/2 and MMP-9 and hPDLC migration were analysized. Results In wound healing tests, the migration of hPDLC exposed to 10% or 20%-cyclic strain at 0. 1 Hz-frequency for 6 hours was not apparent but became significantly different for 24 hours (P <0. 05) compared to control. Furthermore, the 20%-elongation magnitude of cyclic strain had more remarkable effect on migration of hPDLC than 10%-elongation magnitude at 24 hours-duration(P <0. 05). Cyclic strain obviously increased the expression of MMP-9w in hPDLC(P <0. 05). PD98059 could repress not only the activation of p-ERK1/2 but also the expression of MMP-9 induced by eyclic strain in hPDLC. The migration of hPDLC enhanced by cyclic strain was repressed by DOX or PD98059 in wound healing tests. Conclusions Cyclic strain promotes the migration of hPDLC through activating ERK signaling pathway and inducing the expression of MMP-9.