Cell Biological Responses of Human Periodontium Derived Cells by Mechanical Stress

牙周膜位于牙齿和牙槽骨之间，起缓冲咬合和咀嚼等机械应力的作用，而机械应力会影响牙周代谢。本研究旨在探讨机械牵张对<35>人牙周膜成纤维细胞（HPLF）中β-硫代甲硫氨酸（β-S-Met）标记蛋白水平及骨粘连蛋白（osteonectin，ON）合成和基因表达的影响。在Flexercell菌株单元上的Flex I，I型胶原包被的柔性底培养板上培养HPLF。将HPLF在3 ℃（表面积增加7%、18%和24%）下弯曲3个周期（10秒应变和10秒松弛）1、3和5天后，将标记有大分子的培养基上样<35>到IEC-DEAE柱上，用0-0.5M NaCl的PBS溶液（-）洗脱，进行SDS-PAGE（5- 15%凝胶），获得放射自显影图。与非强制降解对照相比，拉伸增加了200 - 100以及70和30 kDa之间的几个放射性标记条带，并转移至离子交换色谱的中性部分。洗脱位置的改变提示可能存在两种可能性，即磷酸化和酸性糖基化等翻译后修饰的减少或修饰物的酶降解。ON是骨非胶原蛋白中调控骨矿化和骨重建的蛋白质之一。如通过蛋白质印迹分析检测的，受到机械应力的ON的合成在HPLF中第一天增加了18%，但结果与对7和24%的机械应力的ON的合成相比没有显著差异。这些结果表明，ON可能参与牙周代谢的机械应力的反应。

Periodontal ligament lies between tooth and alveolar bone and functions as a cushion mitigating mechanical stress such as occlusion and mastication, and the mechanical stress may affect the periodontal metabolism. The object of this study was to demonstrate the effect the mechanical stretching on the level of ^<35>S-Met labeled proteins and the synthesis and the gene expression of osteonectin (ON) in human periodontal ligament-derived fibroblasts (HPLF). HPLF were cultured on Flex I,type I collagen coated flexible-bottomed culture plates on a Flexercell strain unit. HPLF were flexed at 3 cycles (10 sec strain and 10 sec relaxation) at 3 (7,18 and 24% increase in surface area) for 1,3 and 5 days).After macromolecules in media labeled with ^<35>S-Met applied to IEC-DEAE column eluted with 0-0.5M NaCl in PBS (-), SDS-PAGE (5-15%gel) were performed and autoradiograms were obtained. The application of stretching increased of several radiolabeled bands between 200 to 100 and 70 and 30 kDa compared with the non-stress control were shifted to the neutral fraction of of ion-exchange chromatography. The shift of elution position suggested that there may be two possibilities which are the decrease of posttranslational modification such as phosphorylation and acidic glycosylation or enzymatic degradation of modifications.We have focused to ON which has established as one of bone non-collagenous proteins regulating bone mineralization and remodeling. The synthesis of ON subjected to mechanical stress, as examined by Western blotting analysis were increased in HPLF by 18% for the first day, but results were not significantly different as compared to the synthesis of ON to mechanical stress of 7 and 24%. These findings suggest that ON may be involved in periodontal metabolism in response to the mechanical stress.