周期性张应力对成纤维细胞凋亡的影响及其调控机制
摘要
目的：牙周膜成纤维细胞是牙周组织最主要的细胞成分,在功能上也是最重要的细胞,其组织结构和功能可以随外界不同刺激的改变而发生适应性改建。成纤维细胞的增殖、分化及其凋亡在其改建过程中相互影响且缺一不可。因此任何影响成纤维细胞增殖、分化及其凋亡的因素都能导致牙周支持组织的破坏。本研究以体外培养的成纤维细胞为研究对象,在成功构建成纤维细胞体外培养一力学刺激模型的基础上,采用Hoechst 33258细胞染色、TUNEL染色、流式细胞仪及Caspase-3活性检测等方法探讨周期性张应力对成纤维细胞凋亡的影响,阐明Caspase-3在应力介导的牙周膜成纤维细胞凋亡中的作用,明确应力、成纤维细胞凋亡、Caspase-3三者之间的关系,为全面阐明咬合创伤的发病机理和寻找干预治疗的关键靶点提供新思路和理论依据。方法：构建成纤维细胞体外培养一生物力学刺激模型,采用多通道细胞牵张应力加载系统,分别对待测细胞动态加载0h、6h、12h、24h的张应力,其中0h为对照组。实验组细胞的受力值为15%,频率为10cycles/分,每一循环包括3-s stretch/3-s relaxation。采用Hoechst 33258染色及TUNEL染色观察细胞凋亡情况,以流式细胞仪分析细胞周期,同时检测Caspase-3的活性。结果：Hoechst 33258细胞染色结果显示,对照组的细胞核为弥散均匀的圆形或椭圆形荧光,实验组的细胞核或细胞质内可见致密浓染的颗粒、新月体或环状荧光。TUNEL染色结果显示,实验组较对照组的细胞核固缩,边界清楚,细胞浆淡染,染色质呈特异的棕黄色着染。流式细胞仪实验结果显示,成纤维细胞的细胞周期随着加力时间的延长发生了变化,实验组的细胞周期较对照组显著延长。6h组,12h组加力的时间与细胞周期的延长呈正相关,且加力12h时达到高峰。然而,24h组的细胞周期却出现了缩短。加力6h组,12h组的Caspase-3活性明显高于对照组,而加力24h组的Caspase-3活性明显低于12h组但高于对照组。本实验结果提示周期性张应力可加速成纤维细胞的凋亡,在12h内加力时间越长,早期凋亡越明显。结论：周期性张应力能直接影响成纤维细胞的细胞周期；张应力12h时影响成纤维细胞凋亡的作用最为显著。Caspase3活性与张应力对细胞周期及其凋亡的影响密切相关。
Objective:To study the cyclic tensile stress on apoptosis in fibroblasts and its mechanism. Methods:Cyclic tensile stress was applied on the fibroblasts respectively for 0h,6h,12h,24h, morphological changes of cells were observed in using Hoechst 33258,so as to flow cytometry analysis of cell cycle,while detection of Caspase-3 activity. Results:The results of Hoechst 33258 showed that the nucleus for the group without afterburner dispersed uniformly round or oval-shaped fluorescent, while the afterburner groups can be seen within the nucleus or cytoplasm, dense stain particles, crescent or ring fluorescence, nuclear fast shrinkage, some slices of nuclear fragmentation can be seen.Flow cytometry analysis showed that fibroblast cell cycle with the afterburner with the extension of time has changed. Compared with non-afterburner, afterburner group significantly prolonged the cell cycle.6h,12h group with the afterburner time prolonged,the cell cycle also be extended to the 12h peak.24h group also appeared to shorten the cell cycle.Detection of Caspase-3 activity can be seen 6h group,12h group of Caspase-3 activity was significantly higher than that without afterburner group, while the 24h group of Caspase-3 activity was significantly lower than 12h but higher than the group without afterburner. That cyclical tensile stress can induce apoptosis in fibroblasts, within 12 hours afterburner longer the more obvious early apoptosis.Conclusion: Cyclical tensile stress can induce fibroblast apoptosis,can change the cell cycle, and Caspase3 involved in this process.
Objective:To study the cyclic tensile stress on apoptosis in fibroblasts and its mechanism. Methods:Cyclic tensile stress was applied on the fibroblasts respectively for 0h,6h,12h,24h, morphological changes of cells were observed in using Hoechst 33258,so as to flow cytometry analysis of cell cycle,while detection of Caspase-3 activity. Results:The results of Hoechst 33258 showed that the nucleus for the group without afterburner dispersed uniformly round or oval-shaped fluorescent, while the afterburner groups can be seen within the nucleus or cytoplasm, dense stain particles, crescent or ring fluorescence, nuclear fast shrinkage, some slices of nuclear fragmentation can be seen.Flow cytometry analysis showed that fibroblast cell cycle with the afterburner with the extension of time has changed. Compared with non-afterburner, afterburner group significantly prolonged the cell cycle.6h,12h group with the afterburner time prolonged,the cell cycle also be extended to the 12h peak.24h group also appeared to shorten the cell cycle.Detection of Caspase-3 activity can be seen 6h group,12h group of Caspase-3 activity was significantly higher than that without afterburner group, while the 24h group of Caspase-3 activity was significantly lower than 12h but higher than the group without afterburner. That cyclical tensile stress can induce apoptosis in fibroblasts, within 12 hours afterburner longer the more obvious early apoptosis.Conclusion: Cyclical tensile stress can induce fibroblast apoptosis,can change the cell cycle, and Caspase3 involved in this process.
引文
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4 Tsuji K, Uno K, Zhang GX, et al. J Bone Miner Metab,2004,22(2):94-103.
5 Siddhivarn C, Banes A, Champagne C, et al. J Periodontal Res,2006,41(2):92-100.
6 Carano A, Siciliani G. Effects of continuous and intermittent forces on human fibroblasts in vitro[J].Eur J Orthod,1996,18(10):19-26.
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8 彭庭莉,杨军,周继祥.不同张应力对体外培养的人牙周膜成纤维细胞形态和纤维型肌动蛋白改变的影响[J].第三军医大学学报,2005,27(9)：898-900.
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