陳羿貞臺灣大學:臨床牙醫學研究所黃明彥Huang, Ming-YenMing-YenHuang2010-05-262018-07-092010-05-262018-07-092009U0001-2008200915592500http://ntur.lib.ntu.edu.tw//handle/246246/184182牙周韌帶相對於其他組織來說更新較為快速，所以可以快速適應機械力量的刺激，例如矯正牙齒移動的過程中，受力後牙齒隨即將刺激傳導至牙周韌帶，牙周韌帶如果沒有重塑的能力，矯正的治療不可能達成。周韌帶包括了多種細胞如纖維母細胞、造骨細胞、蝕骨細胞、牙釉質母細胞以及細胞外間質，細胞外間質的主要成分為膠原蛋白。膠原蛋白形成牙周韌帶纖維連結牙根表面的牙釉質以及齒槽骨，維持牙周韌帶的寬度以提供立體的空間以利細胞的生長。了幫助牙齒周圍齒槽骨的重塑，細胞外間質的主要成分--膠原蛋白，也會不斷的被分解破壞再重新形成穩定的結構。離胺基氧化酶會催化膠原蛋白共價交聯的過程以形成成熟不可溶的細胞外間質成份。另一方面基質金屬蛋白酶家族以及其組織抑制物的相對活性則扮演將膠原蛋白水解代謝的主要角色，此二者共同調控了牙周組織細胞外間質的恒定。研究目的主要是探討以Flexercell® strain unit給予3%、10%週期性張力於培養在第一型膠原蛋白膜的人類牙周韌帶纖維母細胞，觀察調控膠原蛋白穩定以及促進其分解之酵素(離胺基氧化酶及基質金屬蛋白酶)的表現趨勢。主要是著重在細胞內以及細胞外釋放至培養液的蛋白質層面的探討。研究結果顯示離胺基氧化酶會受3%週期性張力影響上升，張力去除後則表現下降，同時，細胞培養液中也可以測到受張力刺激而表現上升的離胺基氧化酶表現。但是10%張力刺激並未產生更明顯的反應。另一方面，細胞內及培養液中基質金屬蛋白酶-2(MMP-2)在受到3%以及10%週期性張力刺激後都會有表現增加的變化，但細胞內對兩種張力大小的反應差異不大，在培養液中，10%張力刺激後(MMP-2)表現被促進的情況較為明顯。本研究未檢測出細胞中基質金屬蛋白酶-9(MMP-9)的表現，但在培養液中可測到其微量表現且受週期性張力刺激後表現增加，至於對3%與10%張力之反應程度，也是以10%張力刺激之效應較明顯。 總結而言，人類牙周韌帶細胞中離胺基氧化酶以及基質金屬蛋白酶-2，培養液中離胺基氧化酶以及基質金屬蛋白酶-2、-9，都會受到週期性張力刺激而表現增加。顯示機械張力不只是可以直接調控細胞外間質膠原纖維成分的表現，也可能透過調控離胺基氧化酶與基質金屬蛋白酶，而影響細胞間質穩定及代謝的過程。The periodontal ligament (PDL) has a relatively high turnover rate and can therefore quickly adapt to mechanical forces, application during orthodontic tooth movement. PDL and its surrounding tissue are composed of fibroblasts, osteoblasts, cementoblasts, epithelial cells, endothelial cells, and osteoclasts, as well as the extracellular matrix (ECM), of which the major components are the type I and III collagens. Collagens connect the alveolar bone and cementum on the surface of the tooth root, while maintaining the PDL space and providing a three-dimensional scaffold for those cells around the PDL. esides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Lysyl oxidase(LOX) catalyses the cross-linking of fibrillar molecule of type I collagen into mature insoluble extracellular matrix. On the other hand，matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), acting in a co-ordinated fashion to degrade collagen and to regulate the remodeling of periodontal tissues. he purpose of our study was to investigate expression of LOX，MMP-2，and MMP-9，which involve in the process of collagen synthesis，stabilization and degradation in the human periodontal fibroblasts subjected to mechanical stretch. Flexercell® strain unit was used to transmit 0.5 Hz, 3% and 10% equibiaxial tensile force to human periodontal fibroblasts cultured on Type I collagen coated silicone membrane. The biological response was assessed at protein levels. ur results revealed that the expression of LOX enzyme was up-regulated in the periodontal fibroblasts subjected to 3% cyclic mechanical stretch for 24 hours or 48 hours. Meanwhile the increased level of LOX was also noted in the culture medium. These responses diminished after the cells rested for another 48 hours. Compared to 3% stretch, 10% stretch did not produce more significant effect. After being subjected to 3% and 10% mechanical stretch, the levels of MMP-2，the enzyme involved in collagen catabolism，was significantly increased both in the cell layer and in culture medium. The effect of both stretch group were similar in cell layer but the effect of 10% stretch group was more obvious in culture medium. Contrary to MMP-2，the expression of MMP-9 was not noted for cell layer. Possibly, its level was too low to be detected in our system. However，MMP-9 was detected in the culture medium. The level of MMP-9 was induced by 3% and 10% cyclic mechanical stretch，and the effect was more obvious in 10% stretch group. Our data demonstrated that mechanical loading conditions that generated tensional force resulted in an increase in extracellular matrix synthetic events and also in matrix degradative events. The higher levels of LOX，MMP-2，and MMP-9 seen in the periodontal cellular responses to mechanical tensional force may be critically involved in the delicate process regulating the mechanically-induced ECM remodeling of periodontal ligament.目 錄次錄……………………………………………………………………………….…...I次目錄………………………………………………………...…….......................III次目錄………………………………………………………...…….......................III文摘要……………………………………………………………...........................V文摘要………………………………………………………………....................VII一章 緒論……………………………………………………………………….....1二章 文獻回顧.........................................................................................................3三章 實驗目的.......................................................................................................16四章 材料與方法...................................................................................................17五章 結果...............................................................................................................24六章 討論...............................................................................................................28七章 結論...............................................................................................................37八章 未來展望.......................................................................................................39圖..............................................................................................................................40考文獻......................................................................................................................58文索引次一章 緒論 1二章 文獻回顧 3一節 生物力學環境與組織適應 3二節 牙周韌帶的生物角色 3.1 牙周韌帶的功能 4.2 牙周韌帶細胞內與胞外間質組成 4三節 牙齒移動的生物學原理 5.1齒槽骨的可塑性： 6.2牙骨質的抗壓性： 6.3牙周膜內環境的穩定性： 6.4牙齒受力後的組織重塑： 6四節 機械力量與體外實驗模式設計 7五節 從分子生物學觀點來看矯正力量引起的牙齒移動與組織重塑 8六節 與膠原蛋白的平衡相關蛋白之探討 9.1 膠原蛋白的合成 9.2 膠原蛋白的穩定 10.3膠原蛋白的代謝 12三章 實驗目的 16四章 材料與方法 17一節 人類牙周韌帶細胞之培養 17二節 機械張力刺激與細胞培養 18三節 實驗設計 18四節 抽取細胞蛋白質(protein extraction) 19五節 測定蛋白濃度 19六節 西方墨點法(Western blotting) 20七節 酵素圖譜活性分析法(Gelatin Zymography Assay) 22八節 影像分析 23九節 統計分析 23五章 結果 24一節 細胞形態觀察 24二節 西方墨點法(Western blotting)結果 24.1牙周韌帶細胞內的離胺基氧化酶 …. 24.2牙周韌帶細胞培養液的離胺基氧化酶蛋白 24三節 酵素圖譜分析法(Zymography)結果 25.1牙周韌帶細胞內的MMP-2蛋白 25.2牙周韌帶細胞培養液的MMP-2蛋白 26.3牙周韌帶細胞的MMP-9蛋白 26六章 討論 28ㄧ節 受力後細胞形態與排列的改變 28二節 與膠原蛋白穩定相關之蛋白--離胺基氧化酶的表現 29三節 與膠原蛋白分解相關之蛋白--基質金屬蛋白酶(MMPs)的表現 31四節 為何本實驗中人類牙周韌帶細胞內無法表現MMP-9蛋白? 34五節 張力對牙周韌帶細胞的其他影響 35六節 臨床上的應用價值 36七章 結論 37八章 未來展望 39圖 40考文獻 58次目錄次一、總和本篇實驗結果以及蔡芳芳醫師之結果展示..………………………57次目錄次一、膠原蛋白之組成……………………………………………………....40二、膠原蛋白之結構………………………………….. ………….……....40三、離胺基氧化酶以及其相似蛋白族系………….……………………...41四、本實驗設計流程圖與觀察項目……………………………………….42五、牙周韌帶細胞受3%週期性張力刺激後細胞形態之變化…………..43六、牙周韌帶細胞受10%週期性張力刺激後細胞形態之變化………….44七、人類牙周韌帶細胞受3%以及10%週期性張力刺激後，胞內LOX蛋白之表現………………………………………...………………….…45八、人類牙周韌帶細胞受3%週期性張力刺激後，培養液中LOX蛋白之表現………………………...…………………………...……………..46九、人類牙周韌帶細胞受10%週期性張力刺激後，培養液中LOX蛋白之表現………………………...…………………………...…………..46十、人類牙周韌帶細胞受3%週期性張力刺激後，胞內MMP-2蛋白之表...………………………………………………………...………….47十一、人類牙周韌帶細胞受10%週期性張力刺激後，胞內MMP-2蛋白之表現…………………………………………………...…………48十二、人類牙周韌帶細胞受3%週期性張力刺激後，胞內MMP-9蛋白之表現...………………………………………………………...……49十三、人類牙周韌帶細胞受10%週期性張力刺激後，胞內MMP-2蛋白之表現…………………………………………………...….……...50十四、人類牙周韌帶細胞受3%週期性張力刺激後，培養液中MMP-2蛋白之表現...………………………………………………………....51十五、人類牙周韌帶細胞受10%週期性張力刺激後，培養液中MMP-2蛋白之表現…………………………………………………...……52十六、人類牙周韌帶細胞受3%週期性張力刺激後，培養液中MMP-9蛋白之表現...………………………………………………………....53十七、人類牙周韌帶細胞受10%週期性張力刺激後，培養液中MMP-9蛋白之表現………………………………………………………...54十八、人類牙周韌帶細胞受不同強度的週期性張力刺激後，MMP-2分在細胞內以及培養液中、MMP-9在培養液中的表現...………55十九、人類牙周韌帶細胞分別受3%、10%週期性張力刺激後，細胞內及培養液中MMP-2蛋白之表現……………………………….56application/pdf2577046 bytesapplication/pdfen-US牙周韌帶細胞週期性張力刺激膠原蛋白離胺基氧化酶基質金屬蛋白酶-2基質金屬蛋白酶-9periodontal ligamentcyclic tensional forcecollagenlysyl oxidaseMMP-2MMP-9http://ntur.lib.ntu.edu.tw/bitstream/246246/184182/1/ntu-98-R95422017-1.pdf