Cell Biological Responses of Human Periodontium Derived Cells by Mechanical Stress

牙周膜位于牙齿和牙槽骨之间，起着缓冲咬合、咀嚼等机械应力的作用，机械应力可能会影响牙周代谢。本研究旨在探讨机械牵张对人牙周膜成纤维细胞(HPLF)中S蛋氨酸标记蛋白水平及骨连蛋白(ON)合成和基因表达的影响。将HPLF培养在Flexcell单位的Flex I、I型胶原包裹的柔性底培养板上。HPLF在3(比表面积增加7%、18%和24%)下弯曲1、3和5天，共3次循环(10s应变和10s松弛)。将介质中的大分子用^&lt；35&gt；S-蛋氨酸标记后，在IEC-DEAE柱上用0-0.5M氯化钠在PBS(-)中洗脱，进行SDS-PAGE(5-15%凝胶)，获得放射自显影。与非应激对照相比，在200~100 kDa和70 kDa和30 kDa之间拉伸增加的几条放射性标记条带的应用转移到了离子交换层析的中性部分。洗脱位置的改变提示可能存在两种可能性，一种是翻译后修饰的减少，如磷酸化和酸性糖基化，另一种是修饰的酶降解。我们关注的焦点是哪一种已被确立为调控骨矿化和重建的骨非胶原蛋白之一。Western blotting分析表明，在机械应力作用下，HPLF中ON的合成第一天增加了18%，但与7%和24%的机械应力下的ON合成相比，差异无统计学意义。这些发现提示，ON可能参与了牙周组织对机械应力的代谢。

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.