跳到主要內容

臺灣博碩士論文加值系統

(34.204.180.223) 您好!臺灣時間:2021/08/05 16:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:林文心
研究生(外文):Wen-Hsin Lin
論文名稱:凝血酶經由活化TGF-β1誘導人類頰黏膜纖維母細胞結締組織生長因子表現之研究
論文名稱(外文):Thrombin-induced connective tissue growth factor (CTGF/CCN2) expression in human buccal mucosal fibroblasts through TGF-β1 activation
指導教授:郭彥彬郭彥彬引用關係
指導教授(外文):Yen-Ping Kuo
口試委員:張正琪張瑞青
口試委員(外文):Cheng-Chi ChangZuei-Ching Chang
口試日期:2015-07-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:口腔生物科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:51
中文關鍵詞:口腔黏膜下纖維化症凝血酶變形生長因子β茶多酚
外文關鍵詞:OSFThrombinTGF-β1EGCG
相關次數:
  • 被引用被引用:0
  • 點閱點閱:56
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
口腔黏膜下纖維化症 (Oral submucous fibrosis, OSF) 是一種慢性發炎的口腔黏膜病變,臨床症狀為口腔黏膜僵硬、張口、吞嚥以及說話困難。過去研究研究發現嚼食檳榔與 OSF 的產生密切相關。檳榔鹼 (arecoline)、變形生長因子β (TGF-β1)、結締組織生長因子 (CCN2)、微創傷在OSF致病機轉扮演重要角色。先前本實驗室發現,凝血酶在口腔頰黏膜纖維母細胞 (human buccal mucosal fibroblasts, BMFs) 可透過 PAR1、ROS、ASK1、JNK誘導 CCN2 表現;而 TGF-β1 則透過 ALK5、JNK 或 p38 路徑誘導 CCN2 表現,而兩者的 CCN2 表現皆可被茶多酚 (EGCG) 抑制。本研究進一步探討在 BMFs 中 Thrombin 是否會經由TGF-β1誘導頰黏膜纖維母細胞 CCN2 表現及其機轉。
我們首先發現Thrombin可誘導BMFs p-Smad3 的表現。進一步發現TGF-β1 中和抗體、ALK5抑制劑 SB431542、Smad3 抑制劑 SIS3可抑制 Thrombin 誘導的 CCN2 表現,表示 Thrombin 誘導 CCN2 的表現路徑中間有 TGF-β1 訊息路徑的參與。前處理 RGD blocking-peptide 和 integrin 中和抗體亦可以抑制 Thrombin 誘導的活化態 TGF-β1、p-Smad3、以及 CCN2 的表現,顯示 Thrombin 活化 TGF-β1 訊息路徑的過程中有 integrin 的參與;而前處理 ROCK 抑制劑 Y27632、Actin 聚合抑制劑 Blebbistatin 都可以抑制 Thrombin 誘導的活化態 TGF-β1、p-Smad3、以及 CCN2 的表現。顯示在 BMFs 中,Thrombin 可能透過 PAR1/ROCK/Actin polymerization/integrin 訊息路徑調控 TGF-β1 訊息路徑來誘導 CCN2 表現。另外我們亦發現 EGCG 可抑制 Thrombin 誘導的活化態 TGF-β1。EGCG可藉由抑制活化態 TGF-β1來達到抑制或治療口腔黏膜下纖維化症。


Oral submucous fibrosis (OSF) is a chronic inflammatory disease. Its clinical sign and symptoms include stiffness of oral mucosa, difficulty in mouth opening, swallowing and speech. The regular use of areca nut (AN) is the major etiological factor of OSF. Previous study indicated that arecoline, TGF-β1 and microtrauma play an important role in the pathogenesis of OSF. We previously showed TGF-β1 induced CCN2 through ALK5/JNK p38 pathway in human buccal mucosal fibroblasts (BMFs). Thrombin induced CCN2 expression through PAR1/ROS/ASK1/JNK pathway in BMFs. Both of them can be inhibited by epigallocatechin-3-gallate (EGCG). The aim of this study was to investigate whether thrombin induced CCN2 expression in BMFs is via TGF-β1 activation.
We first found thrombin induced Smad3 activation in BMFs. Pretreatment with TGF-β1 neutralizing antibody, ALK5 inhibitor SB431542 and Smad3 inhibitor SIS3 significantly reduced thrombin induced CCN2 expression. These results indicate that thrombin-induced CCN2 expression in BMFs is via TGF-β1-dependent pathway. Pretreatment with RGD blocking-peptide and integrin neutralizing antibodies significantly reduced thrombin induced activated TGF-β1 level, p-Smad3 and CCN2, suggest that integrin is involved in thrombin activate TGF-β1 pathway. Furthermore, ROCK inhibitor Y27632, actin/myosin destabilizing agent blebbistatin significantly reduced thrombin induced activated TGF-β1 level, p-Smad3 and CCN2. In addition, EGCG completely inhibited thrombin-induced activated TGF-β1 level. These results indicated that thrombin-induced CCN2 expression is via TGF-β1 pathway and mediated by PAR1/ROCK/Actin polymerization/integrin cascade.


致謝----------------------------------------------------------------------------------------------Ⅰ
中文摘要----------------------------------------------------------------------------------------Ⅱ
Abstract------------------------------------------------------------------------------------------Ⅲ
目錄---------------------------------------------------------------Ⅳ
導論------------------------------------------------------------------------------------------------1
第一節 口腔黏膜下纖維化----------------------------------------------------------------1
1-1 口腔黏膜下纖維化------------------------------------------------------------------1
1-2 口腔黏膜下纖維化的流行病學---------------------------------------------------1
1-3 口腔黏膜下纖維化的致病機轉---------------------------------------------------2
1-4 口腔黏膜下纖維化的治療---------------------------------------------------------3
第二節 口腔黏膜下纖維化症的分子機轉----------------------------------------------4
2-1 凝血酶 (Thrombin) 及 TGF-β1 誘導CCN2 表現機轉---------------------4
2-2 檳榔鹼 (Arecoline) 及 TGF-β1 訊息路徑間的交互作用-------------------4
2-3 G蛋白偶合受體 (GPCR) 的轉活化作用----------------------------------------6
2-4 不同器官的纖維化疾病中凝血酶 (Thrombin) 活化 TGF-β1 訊息路徑的方式-------------------------------------------------------------------------------------------7
第三節 茶多酚 (EGCG) -------------------------------------------------------------------9
實驗目的----------------------------------------------------------------------------------------11
材料與方法-------------------------------------------------------------------------------------12
第一節 細胞株與細胞培養---------------------------------------------------------------12
第二節 藥物處理---------------------------------------------------------------------------12
第三節 西方墨點法------------------------------------------------------------------------15
第四節 定量聚合脢連鎖反應------------------------------------------------------------17
第五節 酵素連結免疫吸附分析法------------------------------------------------------20
結果----------------------------------------------------------------------------------------------22
Thrombin誘導口腔頰黏膜中CCN2的表現中間有TGF-β1 訊息路徑參與-----22
Thrombin透過轉活化方式影響TGF-β1 訊息路徑誘導CCN2表現--------------22
Thrombin經由PAR1 活化 TGF-β1 訊息路----------------------------------------23
Thrombin活化TGF-β1 訊息路徑的過程中有αvβ1、αvβ3 和 αvβ5 integrin參與-------------------------------------------------------------------------------------------------23
Thrombin透過 Src、RhoA/ROCK 以及 action polymerization 影響αvβ1、αvβ3和αvβ5 integrin 而活化 TGF-β1 訊息路徑----------------------------------------------25
EGCG 可抑制 Thrombin 活化 TGF-β1 訊息路徑的步驟------------------------25
討論----------------------------------------------------------------------------------------------27
圖與表-------------------------------------------------------------------------------------------31
Referances---------------------------------------------------------------------------------------46


Asano, Y., et al. (2006). "Involvement of alphavbeta5 integrin in the establishment of autocrine TGF-beta signaling in dermal fibroblasts derived from localized scleroderma." J Invest Dermatol 126(8): 1761-1769.

Auluck, A., et al. (2008). "Oral submucous fibrosis, a clinically benign but potentially malignant disease: report of 3 cases and review of the literature." J Can Dent Assoc 74(8): 735-740.

Burch, M. L., et al. (2010). "Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type I receptor." J Biol Chem 285(35): 26798-26805.

Burch, M. L., et al. (2013). "Thrombin-mediated proteoglycan synthesis utilizes both protein-tyrosine kinase and serine/threonine kinase receptor transactivation in vascular smooth muscle cells." J Biol Chem 288(10): 7410-7419.

Burch, M. L., et al. (2012). "G protein coupled receptor transactivation: extending the paradigm to include serine/threonine kinase receptors." Int J Biochem Cell Biol 44(5): 722-727.

Cai, Y., et al. (2013). "EGCG inhibits CTGF expression via blocking NF-kappaB activation in cardiac fibroblast." Phytomedicine 20(2): 106-113.

Calderwood, D. A. (2004). "Integrin activation." Journal of Cell Science.

Calderwood, D. S. H. a. D. A. (2009). "Integrin signalling at a glance." Journal of Cell Science 122, 1472

Chang, J. Z., et al. (2012). "Thrombin-stimulated connective tissue growth factor (CTGF/CCN2) production in human buccal mucosal fibroblasts: Inhibition by epigallocatechin-3-gallate." Head Neck 34(8): 1089-1094.

Chang, J. Z., et al. (2013). "EGCG blocks TGFbeta1-induced CCN2 by suppressing JNK and p38 in buccal fibroblasts." Clin Oral Investig 17(2): 455-461.

Chang, Y. C., et al. (2002). "Increased tissue inhibitor of metalloproteinase-1 expression and inhibition of gelatinase A activity in buccal mucosal fibroblasts by arecoline as possible mechanisms for oral submucous fibrosis." Oral Oncol 38(2): 195-200.

Chen, D., et al. (2008). "Tea polyphenols, their biological effects and potential molecular targets." Histology and Histopathology 23(4): 487-496.

Chen, N., et al. (2009). "Green tea, black tea, and epigallocatechin modify body composition, improve glucose tolerance, and differentially alter metabolic gene expression in rats fed a high-fat diet." Nutr Res 29(11): 784-793.

Chiang, C. P., R. P. Hsieh, et al. (2002). "High incidence of autoantibodies in tiwainese patients with oral submucous fibrosis." J Oral Pathol Med.

de Mejia, E. G., et al. (2009). "Bioactive components of tea: cancer, inflammation and behavior." Brain Behav Immun 23(6): 721-731.

Deng, Y. T., et al. (2009). "Arecoline-stimulated connective tissue growth factor production in human buccal mucosal fibroblasts: Modulation by curcumin." Oral Oncol 45(9): e99-e105.

DENNIS A. RICUPERO, D. C. R., PING-PING KUANG, and A. R. H. G. CHRISTINE F. POLIKS (1999). "Regulation of connective tissue growth factor expression by prostaglandin E2."

Friedman, S. L., et al. (2013). "Therapy for fibrotic diseases: nearing the starting line." Sci Transl Med 5(167): 167sr161.

Goodwin, A. and G. Jenkins (2009). "Role of integrin-mediated TGFbeta activation in the pathogenesis of pulmonary fibrosis." Biochem Soc Trans 37(Pt 4): 849-854.

Guo, F., et al. (2011). "Mechanical tension increases CCN2/CTGF expression and proliferation in gingival fibroblasts via a TGFbeta-dependent mechanism." PLoS One 6(5): e19756.

Hasegawa, R., et al. (1995). "Preventive effects of green tea against liver oxidative DNA damage and hepatotoxicity in rats treated with 2-nitropropane." Food Chem Toxicol 33(11): 961-970.

Higdon, J. V. and B. Frei (2003). "Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions." Crit Rev Food Sci Nutr 43(1): 89-143.

Hsin-Ming Chen, Y.-S. K., Mark Yen-Ping Kuo, Chun-Pin Chiaung (2004). "HLA typing in tiwainese patients with oral submucous fibrosis." J Oral Pathol Med.

Imran Khan, N. K., Ila Pant, Sivakrishna Narra, Paturu Kondaiah* (2012). "Activation of TGF-b Pathway by Areca Nut Constituents: A Possible Cause of Oral Submucous Fibrosis." PLoS One 7((12): e51806.).

Ito, Y., et al. (1998). "Expression of connective tissue growth factor in human renal fibrosis." Kidney Int 53(4): 853-861.

Jenkins, R. G., et al. (2006). "Ligation of protease-activated receptor 1 enhances alpha(v)beta6 integrin-dependent TGF-beta activation and promotes acute lung injury." J Clin Invest 116(6): 1606-1614.

Jens J. Pindborg, D. D. S., Dr. Odont., Copenhage11, Denmark, and and M. X. Sntyuzmti M. Sirsat, Ph.D., Bombay, India (1966). Oral submucous fibrosis

Jeremy T. Allen, R. A. K., Claire A. Bloor, and Monica A. Spiteri (1999). "Enhanced Insulin-Like Growth Factor Binding Protein–Related Protein 2 (Connective Tissue Growth Factor) Expression in Patients with Idiopathic Pulmonary Fibrosis and Pulmonary Sarcoidosis." Am. J. Respir. Cell Mol. Biol. 21: 693–700.

Kawai, K., et al. (2003). "Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding." Journal of Allergy and Clinical Immunology 112(5): 951-957.

Leask, A., et al. (2001). "The control of ccn2 (ctgf) gene expression in normal and scleroderma fibroblasts." Mol Pathol 54(3): 180-183.

Little, P. J. (2013). "GPCR responses in vascular smooth muscle can occur predominantly through dual transactivation of kinase receptors and not classical Galphaq protein signalling pathways." Life Sci 92(20-21): 951-956.

Little, P. J., et al. (2011). "The paradigm of G protein receptor transactivation: a mechanistic definition and novel example." ScientificWorldJournal 11: 709-714.

Luttrell, D. K. and L. M. Luttrell (2004). "Not so strange bedfellows: G-protein-coupled receptors and Src family kinases." Oncogene 23(48): 7969-7978.

Mitroulis, I., et al. (2011). "The multivalent activity of the tissue factor-thrombin pathway in thrombotic and non-thrombotic disorders as a target for therapeutic intervention." Expert Opinion on Therapeutic Targets 15(1): 75-89.

Moutasim, K. A., et al. (2011). "Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis." J Pathol 223(3): 366-377.

Murakami, C., et al. (2002). "Effect of tea catechins on cellular lipid peroxidation and cytotoxicity in HepG2 cells." Biosci Biotechnol Biochem 66(7): 1559-1562.

Murti, P. R., et al. (1995). "Etiology of oral submucous fibrosis with special reference to the role of areca nut chewing." J Oral Pathol Med 24(4): 145-152.

Nagle, D. G., et al. (2006). "Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives." Phytochemistry 67(17): 1849-1855.

Niu, G. and X. Y. Chen (2011). "Why Integrin as a Primary Target for Imaging and Therapy." Theranostics 1: 30-47.

Paradis, V., et al. (1999). "Expression of connective tissue growth factor in experimental rat and human liver fibrosis." Hepatology 30(4): 968-976.

Prince, P. T. D. a. P. S. M. (2009). "Preventive Effect of (−)Epigallocatechin Gallate on Lipids, Lipoproteins, and Enzymes of Lipid Metabolism in Isoproterenol-Induced Myocardial Infarction in Rats." J BIOCHEM MOLECULAR TOXICOLOGY 23(DEVIKA AND STANELY MAINZEN PRINCE).

Qin, S., et al. (2013). "Functional characterization of ferret CCL20 and CCR6 and identification of chemotactic inhibitors." Cytokine 61(3): 924-932.

Rajalalitha, P. and S. Vali (2005). "Molecular pathogenesis of oral submucous fibrosis--a collagen metabolic disorder." J Oral Pathol Med 34(6): 321-328.

Rajendran, R. (1994). "Oral submucous fibrosis: etiology, pathogenesis, and future research." Bull World Health Organ 72(6): 985-996.

Reichart, P. A. and H. P. Philipsen (1998). "Betel chewer''s mucosa - a review." Journal of Oral Pathology & Medicine 27(6): 239-242.

Sarrazy, V., et al. (2014). "Integrins alphavbeta5 and alphavbeta3 promote latent TGF-beta1 activation by human cardiac fibroblast contraction." Cardiovasc Res 102(3): 407-417.

Shattil, S. J., et al. (2010). "The final steps of integrin activation: the end game." Nat Rev Mol Cell Biol 11(4): 288-300.

Shen, B., et al. (2012). "Inside-out, outside-in, and inside-outside-in: G protein signaling in integrin-mediated cell adhesion, spreading, and retraction." Curr Opin Cell Biol 24(5): 600-606.

Shen, B., et al. (2013). "A directional switch of integrin signalling and a new anti-thrombotic strategy." Nature 503(7474): 131-135.

Sheng, R., et al. (2010). "Epigallocatechin gallate protects H9c2 cardiomyoblasts against hydrogen dioxides- induced apoptosis and telomere attrition." Eur J Pharmacol 641(2-3): 199-206.

Smith, A., et al. (2010). "Nanolipidic particles improve the bioavailability and alpha-secretase inducing ability of epigallocatechin-3-gallate (EGCG) for the treatment of Alzheimer''s disease." Int J Pharm 389(1-2): 207-212.

Sriram, N., et al. (2009). "Epigallocatechin-3-gallate exhibits anti-fibrotic effect by attenuating bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in rat model pulmonary fibrosis." Chem Biol Interact 180(2): 271-280.

Sullivan, B. P., et al. (2010). "The coagulation system contributes to alphaVbeta6 integrin expression and liver fibrosis induced by cholestasis." Am J Pathol 177(6): 2837-2849.

Tak, J., et al. (2014). "Oral submucous fibrosis: a review article on etiopathogenesis." Kathmandu Univ Med J (KUMJ) 12(46): 153-156.

Tilakaratne, W. M., et al. (2006). "Oral submucous fibrosis: review on aetiology and pathogenesis." Oral Oncol 42(6): 561-568.

Tipoe, G. L., et al. (2010). "Epigallocatechin-3-gallate (EGCG) reduces liver inflammation, oxidative stress and fibrosis in carbon tetrachloride (CCl4)-induced liver injury in mice." Toxicology 273(1-3): 45-52.

Wang, H. J., et al. (2014). "MicroRNA-146a decreases high glucose/thrombin-induced endothelial inflammation by inhibiting NAPDH oxidase 4 expression." Mediators Inflamm 2014: 379537.

Worthington, J. J., et al. (2011). "TGFbeta: a sleeping giant awoken by integrins." Trends Biochem Sci 36(1): 47-54.

Harry C. Dietz. (2015). "One integrin to rule them all ?" Sci Transl Med 7: 288 288fs21

Nilgun I. Reed., et al. (2015) "The avb1 integrin plays a critical in vivo role in tissue fibrosis." Sci Transl Med 7: 288 288ra79



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top