不同等级应力对人炎症牙周膜干细胞分化及细胞骨架重组的研究

The effects of different magnitudes of static mechanical strain on the differentiation and cytoskeletal reorganization of human periodontal ligament stem cells in an inflammatory condition in vitro

目的:探究不同等级静态牵张力(static mechanical strain,SMS)对人炎症牙周膜干细胞(human periodontal ligament stem cells derived from periodontits tissue,h PPDLSCs)分化特性及细胞骨架重组的影响。方法:对体外培养h PPDLSCs分别施加力值为6%,8%,10%,12%,14%的静态牵张力,连续加力12 h(对照组不加力)后,分别通过ALP染色,茜素红染色检测其成骨分化能力;油红O染色检测其成脂能力;倒置显微镜检测细胞形态学改变;免疫荧光染色和共聚焦显微镜观察细胞骨架的改变。Western Blot法检测细胞骨架相关蛋白ROCK1,p-cofilin,RhoGDIα,Rho-A的表达。结果:8%加力组h PPDLSCs镜下矿化结节和脂滴形成明显增多,ALP活性增强;共聚焦显微镜下细胞骨架弹力纤维的形成增多; ROCK1,cofilin,Rho-A细胞骨架正向蛋白表达水平上调,p-cofilin,Rho-GDIα表达水平则明显下降(P<0. 05)。结论:8%静态牵张力明显提高h PPDLSCs增殖分化能力和促进细胞骨架的重组;过大或过小的机械应力均不利于细胞生物学潜能的发挥。

Objective: To investigate the effects of different magnitudes of static mechanical strain( SMS) on the differentiation and cytoskeletal reorganization of human periodontal ligament stem cells derived from periodontitis tissue( h PPDLSCs). Methods: h PPDLSCs were cultured and identified in vitro. After exposed to cyclic SMS loading of 6%,8%,10%,12%,14% respectively at a frequency of 0. 1 HZ for 12 h through a FX-4000 T system applied. Osteogenic and adipogenic differentiation were examined by ALP staining,Alizarin staining,ALP activity test and Oil Red O test. Cytoskeletal reorganization was observed by immunofluorescence staining and confocal microscopy and protein expression was evaluated by Western Blot analysis. Results: SMS with physiological loading of 8% increased the density of mineralized nodules,lipid droplets and the cytoskeletal rearrangement of h PPDLSCs,up-regulated the expression of ROCK1,Rho-A and cofilin,suppressed protein expression levels of Rho-GDIα and p-cofilin. Conclusion: The 8% SMS significantly promote the differentiation ability of h PPDLSCs and recombination of cytoskeleton,however improper mechanical stress may be not conducive to the biological potential of cells.