臺灣博碩士論文加值系統

機械力量刺激對於牙周組織的構造維持及生理平衡是一個重要的調控因子，牙周韌帶細胞會感知這些刺激而作出反應，當中包括細胞外間質的改變(主要成分為膠原蛋白)。維持膠原蛋白含量的動態平衡是藉由膠原蛋白的合成、結構的穩定及分解三個方式的協調運作而達成。膠原蛋白在合成之後需要經由共價交聯(crosslink)，成為較穩定、不可溶的結構，離胺基氧化酶(lysyl oxidase)為參與此一過程的重要酵素，而膠原蛋白的分解主要是透過基質金屬蛋白酶(matrix metalloproteinase，MMPs)。基質金屬蛋白酶的擷抗者為tissue inhibitor of matrix metalloproteinase (TIMPs)，MMPs 與TIMPs之相對活性決定了對基質分解的作用程度。離胺基氧化酶在生物學上扮演著多重的角色，但至今尚無探討其與牙周韌帶細胞膠原蛋白調控相關的研究報告，因此本實驗使用Flexor® cell strain unit施予牙周韌帶細胞3%及10%，頻率0.1赫茲(Hz)的週期性張力，探討機械張力刺激與細胞外基質中膠原蛋白合成、穩定(crosslink)與分解之間的關係，以及離胺基氧化酶、基質金屬蛋白酶所扮演的角色。本實驗結果發現，在受3% 張力24小時、48小時後，與基質合成相關的基因COL-1及LO mRNA表現顯著增加，培養液中膠原蛋白的合成較控制組增加1.1及 2.6倍，離胺基氧化酶的活性則比控制組增加0.6及 2.6倍。 隨著受力時間的增加，細胞的反應也變大，而在刺激消除後48小時則反應變小，且基因的改變與蛋白層級的改變趨勢類似。受10%張力24小時、48小時後，與基質合成相關的基因與蛋白的表現改變不大，3%張力對於調控基質代謝分解的基因(MMP-2、TIMP-1及TIMP-2)影響較小，10%張力則有促進MMP-2基因表現的效果，但其擷抗者TIMP-2的基因表現也有上升情形。根據本實驗的結果，某種程度的機械性張力刺激會使膠原蛋白合成及其相關基因的表現增加，促進人類牙周韌帶細胞的細胞外基質合成，並透過離胺基氧化酶基因與蛋白的活化，使其結構更加穩定。施予細胞張力的時間愈長，細胞的反應似乎愈明顯，但是隨著施力程度增加，細胞的生物反應會有所不同，細胞受力後的反應尚有其他因素控制。細胞對於調控細胞外基質合成與分解的機制相當複雜，了解牙周韌帶細胞對於機械力量刺激的反應，除了有助於了解正常牙周組織生長發育與分化，也可能進一步應用在臨床治療上，例如牙周組織的修復及齒列矯正治療的加速。

Mechanical stimulation is important regulator of structure maintenance and physiological homeostasis in periodontal tissues. Periodontal fibroblasts convert the mechanical signals into various biological responses, including modifying the composition of extracellular matrix(ECM). Collagen is the major ECM component of periodontal fibroblasts and mechanical signals specifically regulate the synthesis and degradation of collagen. After collagen synthesis，crosslink is processed mainly by lysyl oxidase to form more stable, insoluble matrix. The key enzymes involved in degradation of collagen are matrix metalloproteinases (MMPs) family, which interact with tissue inhibitor of matrix metalloproteinases (TIMPs). Lysyl oxidase plays diverse roles in biology, but rare study emphasized on its regulatory effect of collagen level in periodontal cells. The purpose of our study was to investigate the role of this enzyme in the process of collagen synthesis, stabilization and degradation in the periodontal fibroblasts after mechanical stretch. Flexercell® strain unit was used to transmit 0.1Hz, 3% and 10% equibiaxial tensile force to human periodontal fibroblast cultured on 6-well plates with flexible, Type I collagen coated silicone membrane. The biological response was assessed both in mRNA and protein levels. Our results revealed that the expression of COL-1and LO mRNA was up-regulated in the periodontal fibroblasts subjected to 3% cyclic mechanical stretch for 24 hours or 48 hours. The collagen secreted to the medium increased by 1.1 folds and 2.6 folds, and the elevation of lysyl oxidase activity was 0.5 folds and 2.6 folds relative to the control group. These responses diminished after the cells rested for another 48 hours since cessation of the stretch. Compared to 3% stretch, 10% stretch did not produce more significant effect. On the contrary, 10% cyclic stretch did not alter the collagen production and lysyl oxidase activity both in the mRNA and protein levels. After being subjected to 3% mechanical stretch, the mRNA levels of the genes involved in collagen catabolism(MMP-2, TIMP-1, TIMP-2) was not significantly changed in the periodontal fibroblasts. But MMP-2 and TIMP-2 mRNA were up-regulated upon the 10% stretch group. We conclude that cyclic mechanical stretch may enhance production of extracellular matrix in periodontal fibroblast by increasing both gene and protein expression in collagen synthesis. The collagen production is stabilized by the up-regulation in LO mRNA and activation of lysyl oxidase. Since the mechanism of mechanotransduction and regulation in extracellular matrix is complicated, efforts on this field facilitate understanding of the development, differentiation of the periodontium, and the therapeutic potential in the periodontal tissue repair and acceleration of orthodontic tooth movement.

表次目錄…………………………………………………… 111
圖次目錄…………………………………………………… IV
中文摘要 …………………………………………………… Vl
英文摘要 …………………………………………………… Vlll
第一章 引言………………………………………………………………1
一、牙周韌帶的功能………………………………………………………………1
二、牙周韌帶的結構……………………………………………………………... 2
三、機械力量刺激對牙周韌帶結構的影響………………………………………3
四、膠原蛋白的合成………………………………………………………………6
五、膠原蛋白的穩定- Collagen crosslink…………………………………………7
六、膠原蛋白的分解………………………………………………………………10
第二章 實驗目的…………………………………………………………………13
第三章 材料與方法………………………………………………………………14
一、細胞培養…………………………………………………………………..…14
二、張力系統…………………………………………………………………..…14
三、實驗設計…………………………………………………………………..…15
四、膠原蛋白合成之定量……………………………………………………..…15
五、離胺基氧化酶活性測定…………………………………………………..…16
六、半定量-反轉錄-聚合酶連鎖反應…………………………………….…..…17
七、統計分析…………………………………………………………………..…19
第四章 結果…………………………………………………………………………20
一、細胞形態觀察………………………..………………………………………20
二、半定量-反轉錄-聚合酶連鎖反應結果……………………………...………20
三、膠原蛋白合成之定量………………..………………………………………23
四、離胺基氧化酶活性測定…………..…………………………………………24
第五章 討論…………………………………………………………………………25
一、膠原蛋白合成及其相關基因的表現……………………..…………………25
二、離胺基氧化酶基因及其活性的表現……………………..…………………27
三、基質金屬蛋白酶基因的表現……………………………………………..29
四、不同張力大小的結果差異……………………………………..…………31
第六章 結論…………………………………………………………………………33
第七章 未來研究方向………………………………………………………..……34
附錄(圖與表)…………………………………………….…………………………..35
參考文獻…………………………………………………………………….…58

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