臺灣博碩士論文加值系統

Periostin是Matricellular proteins的成員之一，它存在於人體含豐富膠原蛋白的結締組織中，包括心臟瓣膜、肌腱、軟骨膜、骨膜和牙周組織等。研究顯示Periostin基因剔除的老鼠牙周韌帶將無法承受咬合力，牙周組織呈現類似早發性牙周病破壞的情形，可見Periostin在牙周組織因應機械性力量環境變化及結構重塑維持平衡有其重要性。機械力刺激可能透過許多路徑調控人類牙周韌帶細胞Periostin的表現，本研究以 FlexercellR Strain Unit 給予低強度（3%）或高強度（10%）的週期性張力刺激人類牙周韌帶細胞4、8、24及48小時，探討其TGF-β1訊息傳導路徑及Periostin基因和蛋白表現的趨勢; 並且透過加入多種抑制劑以阻斷TGF-β1訊息傳導路徑或細胞骨架動態結構，觀察其對於張力刺激調控Periostin蛋白表現的影響。
本研究結果顯示3%與10%的週期性張力刺激皆會顯著活化人類牙周韌帶細胞TGF-β1訊息路徑，對於Periostin蛋白的表現在初期也都有促進作用，而且10%張力對細胞的刺激效應大於3%張力，但是長時間的10%高張力刺激卻會抑制牙周韌帶細胞表現Periostin蛋白。雖然細胞接受週期性張力刺激後，Periostin蛋白會有初期提升的情形，但Periostin mRNA表現並沒有顯著變化; TGF-β1蛋白表現量亦有初期提升的情形，然而TGF-β1 mRNA表現卻有被向下調控的趨勢。
本研究也證實TGF-β1會刺激牙周韌帶細胞Periostin mRNA的表現; 而施予張力刺激24及48小時下，同時給予TGF-β第一類型受器的阻斷劑（SB431542），結果顯示SB431542會顯著抑制張力刺激促進Periostin和TGF-β1蛋白的表現，但卻不會影響張力刺激Periostin在4小時的促進作用。牙周韌帶細胞承受張力刺激4小時，同時分別給予影響細胞骨架動態結構的抑制劑，包括肌動蛋白聚合抑制劑（Cytochalasin D）、肌動蛋白解聚抑制劑（Jasplakinolide）以及FAK磷酸化抑制劑（Y15），結果皆顯示Periostin蛋白的表現量明顯下降，可見在牙周韌帶細胞接受張力刺激而促成Periostin表現量升高的過程當中，除了TGF-β1訊息活化，細胞骨架動態結構也扮演著重要角色，而且力量刺激經由細胞骨架的傳導可能早於透過TGF-β1訊息活化所引發的作用。
由本研究結果得知，適當的機械力刺激會促進人類牙周韌帶細胞Periostin蛋白表現，無論是透過初期細胞骨架動態結構改變或TGF-β1訊息傳導路徑，皆是細胞因應對外界力量刺激，為維持細胞外基質結構完整與穩定所作出的反應，而健全的細胞骨架動態結構和TGF-β1訊息路徑，在當中皆扮演重要的角色。

Periostin was classified as one of the matricellular proteins predominantly expressed in collagen-rich connective tissues, including heart valves, tendons, perichondrium, periosteum, and periodontal ligament. The periostin knock-out mice showed loss of the tissue integrity of the periodontium in response to mechanical loading, and developed an early-onset periodontal disease-like phenotype. Previous studies suggest that periostin is essential for connective tissue homeostasis and important to maintain the integrity and function of periodontal ligament in respond to mechanical stress. Mechanical strain regulates periostin expression involved of multiple signaling pathways. In the present study, we hypothesized that low-level and high-level tensional force differentially regulates TGF-β1 and periostin expression in human PDL cells. We investigated the TGF-β1 signaling and periostin expression in human PDL cells subjected to low- and high- level cyclic tensional force (3% versus 10% elongation) for 4, 8, 24, and 48 hours in a Flexercell Strain Unit.
Our results revealed that application of 3% or 10% cyclic tensional force activated TGF-β1 signaling pathway and the protein level of periostin in human PDL cells. The stimulatory effect of 10% tensional force was more intensive than that of 3%. However, the high-level tensional force (10%) with long duration (48 hours) inhibited the expression of periostin. Although mechanical stimulation of cyclic tensional force for 4 hours induced an increase in the protein level of periostin, there was no significant change in the mRNA level. The mechanically-induced periostin expression was significantly decreased after the treatment of an inhibitor of TGF-β type I receptor (SB431542) for 24 hours, but not for 4 hours. Furthermore, the inhibitors of actin polymerization (Cytochalasin D), actin depolymerization (Jasplakinolide), or FAK autophosphorylation (Y15), which disrupting cytoskeletal dynamics, decreased mechanically-induced periostin expression at 4 hours. It implied that cytoskeletal dynamics for reorganization of actin filaments preceded the TGF-β1 signaling pathway in the process of mechanotransduction.
In conclusion, the results of our study suggest that different levels of mechanical stress differentially regulate the expression of periostin in PDL cells to ensure the integrity of the periodontium in response to occlusal load. An intact cytoskeletal dynamics and activation of TGF-β1 signaling pathway are essential for mechanically-induced periostin expression in human PDL cells.

口試委員會審定書 II
致謝 III
摘要 IV
Abstract VI
目錄 VIII
圖表目錄 XI
第一章 緒論 1
1-1 牙周韌帶的生物角色 1
1-1-1 牙周韌帶功能 1
1-1-2 牙周韌帶細胞外基質組成 1
1-1-3 Matricellular Proteins 2
1-2 機械力的刺激 3
1-2-1 機械力刺激誘導訊息傳導路徑 3
1-2-2 機械性張力刺激 3
第二章 文獻回顧 4
2-1 Periostin 蛋白之探討 4
2-1-1 Periostin 於人體內扮演的角色 4
2-1-2 Periostin 於牙齒組織中扮演的角色 5
2-2 Periostin與牙周韌帶穩定性的探討 6
2-3 機械性張力刺激與Periostin表現路徑的探討 6
2-3-1 週期性張力刺激經TGF-β1路徑誘發Periostin表現 6
2-3-2 細胞骨架結構及其動態變化對Periositn表現之影響 7
2-3-3 週期性張力刺激對細胞的影響 8
第三章 研究目的與研究假說 9
第四章 實驗材料與方法 10
4-1 人類牙周韌帶細胞培養 10
4-2 張力系統與週期性張力刺激 10
4-3 抑制藥物製備 11
4-3-1 TGF-β1路徑阻斷劑 11
4-3-2 FAK路徑阻斷劑 11
4-3-3 細胞骨架動態結構抑制劑 12
4-4 Periostin及TGF-β1基因的表現 12
4-4-1 RNA萃取 12
4-4-2 反轉錄成cDNA 13
4-4-3 反轉錄聚合酶連鎖反應（Reverse Transcription-Polymerase Chain Reaction，RT-PCR） 13
4-4-4 瓊脂凝膠電泳分析（Agarose gel electrophoresis） 14
4-5 Periostin蛋白及TGF-β1訊息路徑的表現 14
4-5-1 抽取細胞蛋白質 14
4-5-2 測定蛋白濃度 14
4-5-3 西方墨點法（Western blot） 15
4-6 影像分析 16
4-7 統計分析 16
第五章 結果 16
5-1 牙周韌帶細胞形態之觀察 16
5-1-1 3% 週期性張力刺激對牙周韌帶細胞形態之影響 17
5-1-2 10% 週期性張力刺激對牙周韌帶細胞形態之影響 17
5-2 週期性張力刺激對牙周韌帶細胞之Periostin及TGF-β1基因表現的影響 17
5-2-1 TGF-β1誘發牙周韌帶細胞之Periostin及TGF-β1基因表現 17
5-2-2 3% 週期性張力刺激對牙周韌帶細胞之Periostin及TGF-β1基因的影響 18
5-2-3 10% 週期性張力刺激對牙周韌帶細胞之Periostin及TGF-β1基因的影響 18
5-3 週期性張力刺激對牙周韌帶細胞之Periostin蛋白及TGF-β1訊息表現的影響 18
5-3-1 3% 週期性張力刺激對牙周韌帶細胞之Periostin蛋白表現的影響 18
5-3-2 3% 週期性張力刺激對牙周韌帶細胞之TGF-β1蛋白表現的影響 19
5-3-3 3% 週期性張力刺激對牙周韌帶細胞之Smad 2/3, p-Smad 2, p-Smad3蛋白表現的影響 19
5-3-4 10% 週期性張力刺激對牙周韌帶細胞之Periostin蛋白表現的影響 19
5-3-5 10% 週期性張力刺激對牙周韌帶細胞之TGF-β1蛋白表現的影響 20
5-3-6 10% 週期性張力刺激對牙周韌帶細胞之Smad 2/3, p-Smad 2, p-Smad3蛋白表現的影響 20
5-4 抑制劑對牙周韌帶細胞之Periostin蛋白及TGF-β1訊息表現的影響 20
5-4-1 抑制劑對牙周韌帶細胞形態影響之觀察 20
5-4-2 TGF-β1路徑阻斷劑對牙周韌帶細胞之Periostin蛋白及TGF-β1訊息表現的影響 21
5-4-3 細胞骨架動態結構抑制劑對牙周韌帶細胞之Periostin蛋白及TGF-β1訊息表現的影響 21
5-4-4 FAK路徑阻斷劑對牙周韌帶細胞之Periostin相關蛋白的影響 22
第六章 討論 22
6-1 張力刺激對細胞形態與排列的影響 22
6-2 TGF-β1誘發牙周韌帶細胞之Periostin及TGF-β1基因表現 24
6-3 3% 張力刺激對Periostin及TGF-β1基因表現的影響 25
6-4 10% 張力刺激對Periostin及TGF-β1基因表現的影響 26
6-5 3% 張力刺激對Periostin相關蛋白表現之影響 27
6-6 10% 張力刺激對Periostin相關蛋白表現之影響 28
6-7 抑制劑對細胞形態之影響 29
6-8 TGF-β1路徑阻斷藥物對Periostin相關蛋白的影響 30
6-9 細胞骨架動態結構抑制劑對Periostin相關蛋白的影響 30
6-10 FAK路徑阻斷藥物對Periostin相關蛋白的影響 31
第七章 結論 32
第八章 未來研究 33
參考文獻 59

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