- 學位論文
鈣離子通道阻斷劑Nifedipine對於機械張力調控MG-63類骨母細胞表現TGF-β、Periostin、Collagen的影響
Effects of Nifedipine on Mechanical Stress-induced Periostin, TGF-β and Collagen Expression in MG-63 Osteoblastic Cells
摘要
Nifedipine為鈣離子通道抑制劑，臨床上常用於治療高血壓或心絞痛。使用Nifedipine藥物的患者，可能會有牙齦增生之副作用，據統計，好發機率介於15%到83%。動物實驗發現Nifedipine會減少矯正牙齒的移動，此外，也有基礎研究發現牙齦纖維母細胞（gingival fibroblast）當受到Nifedipine刺激時，會增加其膠原蛋白(collagen)的合成。 Periostin是近年來，研究人員在extracellular matrix發現的一種親和性蛋白質，從眾多學者的研究統計發現，Periostin通常容易出現於具有豐富纖維蛋白的纖維軟組織，包括心臟瓣膜，牙周韌帶等組織，參與非常多生理機轉如心肺修復，牙周韌帶重塑，骨生理反應等。研究顯示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不同調控路徑影響人類牙周韌帶細胞Periostin的表現，其中已證實機械力刺激可以通過Transforming frowth factor-β1 (TGF-β1)訊息傳導路徑活化和促進Periostin蛋白的表現。 本研究以 Flexercell® Strain Unit 給予3%的週期性張力刺激人類MG-63類骨母細胞，作用時間為4及24小時，並給予30 ng/ml和100 ng/ml的Nifedipine刺激，探討鈣離子通道阻斷劑Nifedipine對於機械張力調控MG-63類骨母細胞表現TGF-、Periostin、Collagen的影響。 本研究發現Nifedipine作用4小時，即可看出藥物對TGF-β1以及Periostin表現的影響，無論有無週期性張力刺激，皆會促進TGF-β1以及Periostin的表現；藥物以及張力持續作用24小時,受力組的TGF-β1和Periostin表現相較於無受力組是增加的，並且增加的幅度隨著藥物濃度增加而減少。Collagen表現方面，4小時的Nifedipine作用並不如機械性張力的效應，因為無論有無藥物刺激，張力皆可促進COL1A1的表現。持續刺激24小時，機械性張力皆會減少COL1A1的表現，而作用24小時，可以看得出Nifedipine的效應，COL1A1的表現隨著藥物濃度增加而更為減少。 因為Nifedipine是L型鈣離子通道阻斷劑，而細胞的鈣離子通透性之調控種類繁多，因此本研究使用1 mM以及2 mM EGTA鈣離子螯合劑將細胞外鈣離子螯合，對照於Nifedipine的作用，探討兩者之結果是否有相應之處。EGTA作用4小時對3%，0.1Hz週期性張力刺激調控TGF-β1的表現沒有顯著影響(p < 0.05)；持續刺激24小時, 只有2 Mm EGTA在合併週期性張力刺激時，可以促進TGF-β1的表現(p < 0.05)。Periostin方面，EGTA合併張力刺激4小時能夠促進periostin的表現；持續刺激24小時，無論有無藥物作用，Periostin的表現相較於無受力組皆有減少的趨勢。4小時的COL1A1的表現，無論有無EGTA刺激，3%，0.1Hz週期性張力皆可促進COL1A1的表現。持續刺激24小時，無論有無藥物刺激，張力皆會減少COL1A1的表現，並且隨著藥物濃度增加而減少 。 由本研究結果得知，Nifedipine參與週期性張力（3%, 0.1 Hz）對MG-63類骨母細胞表現TGF- β1、Periostin以及Collagen的調控，Nifedipine作用24小時，會負向調控張力所誘導的TGF- β1以及其下游產物periostin的表現。Collagen的分泌增加與齒槽骨的結構穩定有關，機械張力刺激4小時觀察到collagen合成增加，但是刺激24小時，collagen合成減少，而Nifedipine作用使collagen合成的表現更明顯被弱化。
並列摘要
Nifedipine, as known as calcium channel blocker, is clinically be used in the treatment of hypertension or angina. Statistically, patients using nifedipine may have side effects of gingival hyperplasia, with the rate of between 15% to 83%. On the other hand, animal models had proved that nifedipine reduces the orthodontic tooth movement. In addition, studies also found the increase of collagen synthesis in gingival fibroblast in response to the stimulation of nifedipine. Periostin is a similar affinity protein found in recent years by researchers in the extracellular matrix. Periostin is often found in fibrous soft tissue with rich fibrin, including heart valves, periodontal ligaments, and so on. It is involved in a lot of physiologic organs such as cardiopulmonary repair, PDL remodeling, bone physiological response, etc. Previous studies suggested 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. Previous studies revealed that cyclic tensional force can activate and promote the expression of periostin through transforming growth factor-β1 (TGF-β1) signaling pathway. In this study, MG-63 osteoblastic cells was stimulated by a low-intensity (3%) cyclic tensional force for 4 or 24 hours with a Flexercell® Strain Unit and 30 ng / ml or 100 ng / ml nifedipine to investigate effects of nifedipine on mechanical stress-induced TGF-, periostin and collagen expression. Our study found that nifedipine stimulation for 4 hours, with or without 3%, 0.1Hz cyclic tensional force stimulation, promoted the expression of TGF-β1 and periostin, nifedipine effect can be noted within 4 hours. Stimulation of nifedipine and tensional force for 24 hours, expression of TGF-β1 and periostin were increased compared with control group, but the rate of increase decreases as the nifedipine concentraction increases, nifedipine effect are stronger than cyclic tensional force. The expression of COL1A1 was promoted after stimulation of periodic tensional force, with or without nifedipine stimulation. Sustained stimulation for 24 hours, with or without nifedipine stimulation, tensional force reduce the expression of COL1A1, and the rate of decrease increases as the nifedipine concentration increases, nifedipine effect can be noted. 1 mM and 2 mM EGTA calcium chelating agent, which can chelate the extracellular calcium, was used to explore whether the effect on mechanical stress-induced protein expression is same with nifedipine. Only 2 mM EGTA-treated group promotes the expression of TGF-β1 after stimulation of tensional force for 24 hours (p <0.05). Stimulation of EGTA and tensional force for 4 hours promote the expression of periostin; sustained stimulation for 24 hours, with or without drug effects, tensional force increses the expression of periostin. On the other hand, with or without EGTA stimulation, 3%, 0.1Hz periodic tensional force promotes the expression of COL1A1. Sustained stimulation for 24 hours, with or without drug stimulation, tensional force reduces the expression of COL1A1, and the rate of decrease increases as the drug concentration increases. The results of this study show that nifedipine may involve in the regulation of mechanical stress-induced TGF-β1, periostin and collagen expression in MG-63 osteoblastic cells. Nifedipine stimulation for 24 hours down-regulated the expression of mechanical stress-induced TGF-β1 and periostin. The secretion of collagen was related to the structural stability of the alveolar bone. The expression of collagen was increased after 4 hours of tensional force stimulation. However, the expression of collagen was obviously weakened after stimulation of nifedipine and tensional force for 24 hours. In conclusion, our study had shown us that alveolar bone remodeling would be interrupted with usage of nifedipine, thereby affecting the orthodontic tooth movement rate. Further studies are needed to investigate the signal transduction of the nifedipine on regulation of mechanical stress-induced TGF-β1, periostin and collagen expression. Also, animal experiments to clarify and verify our conclusion.