Rho信号通路在周期性张应力诱导人牙周膜细胞骨架重排中的作用

Rho plays a key role in cyclic strain induced cytoskeletal rearrangement of human periodontal ligament cells

目的探讨周期性张应力对人牙周膜细胞(human periodontal ligament cells,hPDLCs)骨架重排的影响及Rho信号通路在其中的作用。方法应用FX-5000T细胞应变加载系统,对体外培养的hPDLCs施加周期性张应力,幅度为10%,频率为0.1 Hz,加载时间为6 h和24 h。以未加载的细胞作为对照组。应用倒置显微镜观察细胞形态变化,细胞免疫荧光染色和共聚焦显微镜观察细胞骨架蛋白F-actin的变化,并应用Western blot检测Rho通路相关蛋白的表达变化。用Rock的特异性抑制剂Y-27632预处理细胞后,在0.1 Hz,10%幅度条件下,加载6 h和24 h,检测细胞骨架蛋白F-actin的变化和Rho信号通路相关蛋白的表达变化。结果与静态组相比,周期性张应力诱导hPDLCs细胞骨架重排差异有统计学意义,Rho信号通路Rock和p-cofilin蛋白表达增加(P<0.05)。Rock的特异性抑制剂Y-27632可以降低Rock(P<0.05)和p-cofilin蛋白表达和细胞骨架重排。结论周期性张应力可促进体外培养的hPDLCs细胞骨架重排,Rho信号通路参与了此过程。

Objective To investigate the impact of cyclic strain on cytoskeletal arrangement of human periodontal ligament cells（ hPDLCs） and the mechanism of Rho signaling pathway plays in it. Methods Ten percent-elongation of cyclic strain at 0. 1 Hz-frequency was applied to hPDLCs for 6 h or 24 h with FX-5000T system. The hPDLCs without cyclic strain under the same conditions were used as the control. The cell morphological changes were examined by phase-contrast microscopy. F-actin reorganization was observed by immunochemistry and confocal microscopy. Protein expression of Rho signaling pathway was analyzed by Western blot analysis. HPDLCs were preincubated with Y-27632,a specific Rock inhibitor,before cyclic strain application. F-actin reorganization was observed by immunochemistry and confocal microscopy,protein expression of Rho signaling pathway was also analyzed by Western blot analysis after 10%-elongation of cyclic strain at 0. 1 Hz-frequency was applied to hPDLCs for 6 h or 24 h. Results Cytoskeletal reorganization was observed after cyclic strain,compared to the static control. The strain also significantly increased Rock and p-cofilin expressions in hPDLCs（ P 0. 05）. Additionally,Y-27632 devreased Rock and p-cofilin protein expressions（ P 0. 05） and cytoskeletal reorganization under cyclic strain. Conclusion Cyclic strain promotes cytoskeletal rearrangement of hPDLCs by Rho signaling pathway.