跳到主要內容

臺灣博碩士論文加值系統

(18.97.14.86) 您好!臺灣時間:2025/02/08 02:51
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:張亞維
研究生(外文):Chang,Yawei
論文名稱:探討Toll-Like Receptor 2 誘導血管平滑肌細胞遷移作用所扮演的角色
論文名稱(外文):Toll-Like Receptor 2 signaling plays a pivotal role in arotic vascular smooth muscle cells migration
指導教授:周慰遠郭呈欽
指導教授(外文):Chou,WeiyuanKuo,Chengchin
口試委員:劉俊揚林秀芳
口試委員(外文):Liou,JunyangYet,Shawfang
口試日期:2011-06-24
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:107
中文關鍵詞:類譯受體平滑肌細胞遷移白細胞介素6
外文關鍵詞:Toll-like receptorsmigrationinterlukine-6
相關次數:
  • 被引用被引用:0
  • 點閱點閱:315
  • 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
類譯受體 ( Toll-like receptors,TLRs ) 為一類模式辨認受體 ( pattern recognition receptor ),可以辨認病源相關分子模式 ( pathogen-associated molecular patterns,PAMPs),像是細菌、原生動物,真菌與病毒,而引發發炎反應,因此TLRs 在先天性免疫反應中扮演者重要的角色。文獻中指出當血管受到傷害及感染時會引發發炎反應並且刺激細胞激素 ( cytokine ) 與化學激素 ( chemokine ) 產生,而這些激素會促進平滑肌細胞從血管中層遷移到內壁,過多的平滑肌細胞遷移並堆疊造成血液流通量變少,最後導致慢性動脈粥狀硬化的發生。現今許多疾病與慢性發炎有關,像是心血管方面疾病動脈粥狀硬化。因此我們想要探討在小鼠動脈平滑肌細胞中,TLR2所引發的訊號傳遞路徑是透過哪些激酶與轉錄因子來調控細胞激素的產生及遷移作用 ( migration ) 的發生。在小鼠動脈平滑肌細胞中,在 TLR2 的配體 Pam3CSK4 刺激下可以促進白細胞介素6 ( interlukine-6,IL-6 ) 表現以及活化 PI3K-AKT、p38 MAPK、ERK1/2 及 JNK。我們進一步探討其訊息傳遞路徑,從 MAPK 及 PI3K-AKT 激酶抑制實驗中發現,當個別加入 PI3K-AKT、p38 MAPK 及 ERK1/2 抑制劑後,以酶聯免疫吸附試驗發現會降低 TLR2 引起 IL-6 表現。接者以野生型及 tlr2 基因剔除小鼠試驗來證明是否會透過 TLR2 影響細胞遷移。結果證明在 TLR2 訊號傳遞路徑下,會誘導小鼠動脈平滑肌細胞遷移,並且利用 IL-6 抗體中和試驗更證明 IL-6 參與 TLR2 誘導動脈平滑肌細胞遷移。以上結果皆指出 TLR 2 可以透過 IL-6 調控平滑肌細胞的遷移,隨後利用激酶抑制劑實驗發現會透過 p38 MAPK、ERK1/2 及 PI3K-AKT 來調控細胞遷移。我們進一步探討發現在 TLR2 訊號傳遞路徑下會活化轉錄因子 NF-κB、CREB 及 ATF2 。而當個別加入 p38 MAPK 及 ERK1/2 抑制劑後會抑制 CREB 磷酸化;加入 JNK 抑制劑後會抑制 ATF2 磷酸化。並且利用電穿孔實驗將 CREB siRNA 送入小鼠動脈平滑肌細胞後會降低 CREB 蛋白質的量,並會抑制 IL-6 表現及細胞遷移作用。綜合以上結論顯示,在小鼠動脈平滑肌細胞中,TLR2 訊號傳遞路徑會透過 p38 MAPK及 ERK1/2 來調控 CREB 活化,當 CREB 被活化後可以更進一步影響 IL-6 表現及細胞遷移作用發生。
Toll-like receptors (TLRs) are pattern recognition receptors that interact with components of pathogens and transformed cells to initiate inflammatory pathway which may exacerbate chronic inflammatory diseases like atherosclerosis. Vascular smooth muscles cells (VSMCs) migration from tunica media into the intima is associated with the development of vascular diseases. The aim of the present study is to investigate the role of TLR2 signaling in VSMCs migration. Boyden chamber assay indicated TLR2 synthetic ligand Pam3CSK4 stimulated mouse VSMC migration. Inhibition of TLR2 signaling by anti-TLR2 antibody or TLR2 knockout suppressed Pam3CSK4-induced VSMCs migration. Upon Pam3CSK4 stimulation, the activity of p38 MAPK, ERK1/2, PI3K/AKT and JNK was induced in VSMCs. Blockade of p38 MAPK, ERK1/2 or PI3K/AKT but not JNK inhibited Pam3CSK4-induced VSMCs migration and IL-6 secretion. Neutralizing anti-IL-6 antibodies blocked Pam3CSK4-induced VSMCs migration. Further studies indicated that IL-6 promoter regulatory elements CREB, NF-κB(p65) and ATF2 were activated in Pam3CSK4-stimulated VSMCs. Blockade of ERK1/2 or p38 MAPK suppressed Pam3CSK4-upregulated phosphorylation level of CREB but not ATF2 and NF-κB(p65). Inhibition of CREB level by expressing small-interfering RNA suppressed not only CERB expression but also Pam3CSK4-induced phosphorylation of CREB and IL-6 production. Moreover, CREB siRNA also suppressed Pam3CSK4-induced VSMC migration. Collectively, the finding suggests that that IL-6 production induced by Pam3CSK4 is mediated by triggering both ERK1/2 and p38 MAPK signaling, resulting in activation of CREB and subsequent CREB-mediated IL-6 production that promotes VSMCs migration.
目錄

一、緒論-------------------------------------------------------------------1
二、實驗材料及訂購公司---------------------------------------------12
三、實驗緩衝液與藥品配置------------------------------------------15
四、實驗方法------------------------------------------------------------23
五、實驗結果------------------------------------------------------------38
六、討論------------------------------------------------------------------52
七、結果圖表------------------------------------------------------------67
八、參考文獻------------------------------------------------------------87

1.Akira, S., K. Takeda, and T. Kaisho. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2:675-680.
2.Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394-397.
3.Lemaitre, B., E. Nicolas, L. Michaut, J. M. Reichhart, and J. A. Hoffmann. 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86:973-983.
4.Yamamoto, M., and K. Takeda. 2010. Current views of toll-like receptor signaling pathways. Gastroenterol Res Pract 2010:240365.
5.Jin, M. S., and J. O. Lee. 2008. Structures of the toll-like receptor family and its ligand complexes. Immunity 29:182-191.
6.Akira, S. 2001. Toll-like receptors and innate immunity. Adv Immunol 78:1-56.
7.Gautam, J. K., Ashish, L. D. Comeau, J. K. Krueger, and M. F. Smith, Jr. 2006. Structural and functional evidence for the role of the TLR2 DD loop in TLR1/TLR2 heterodimerization and signaling. J Biol Chem 281:30132-30142.
8.Dolan, J., K. Walshe, S. Alsbury, K. Hokamp, S. O'Keeffe, T. Okafuji, S. F. Miller, G. Tear, and K. J. Mitchell. 2007. The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns. BMC Genomics 8:320.
9.Slack, J. L., K. Schooley, T. P. Bonnert, J. L. Mitcham, E. E. Qwarnstrom, J. E. Sims, and S. K. Dower. 2000. Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways. J Biol Chem 275:4670-4678.
10.Underhill, D. M., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson, M. Bassetti, and A. Aderem. 1999. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature 401:811-815.
11.Xu, Y., X. Tao, B. Shen, T. Horng, R. Medzhitov, J. L. Manley, and L. Tong. 2000. Structural basis for signal transduction by the Toll/interleukin-1 receptor domains. Nature 408:111-115.
12.Tabeta, K., K. Hoebe, E. M. Janssen, X. Du, P. Georgel, K. Crozat, S. Mudd, N. Mann, S. Sovath, J. Goode, L. Shamel, A. A. Herskovits, D. A. Portnoy, M. Cooke, L. M. Tarantino, T. Wiltshire, B. E. Steinberg, S. Grinstein, and B. Beutler. 2006. The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat Immunol 7:156-164.
13.Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11:373-384.
14.Kang, J. Y., X. Nan, M. S. Jin, S. J. Youn, Y. H. Ryu, S. Mah, S. H. Han, H. Lee, S. G. Paik, and J. O. Lee. 2009. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 31:873-884.
15.Hasan, U., C. Chaffois, C. Gaillard, V. Saulnier, E. Merck, S. Tancredi, C. Guiet, F. Briere, J. Vlach, S. Lebecque, G. Trinchieri, and E. E. Bates. 2005. Human TLR10 is a functional receptor, expressed by B cells and plasmacytoid dendritic cells, which activates gene transcription through MyD88. J Immunol 174:2942-2950.
16.Jin, M. S., S. E. Kim, J. Y. Heo, M. E. Lee, H. M. Kim, S. G. Paik, H. Lee, and J. O. Lee. 2007. Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071-1082.
17.Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith, C. B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci U S A 97:13766-13771.
18.Underhill, D. M., A. Ozinsky, K. D. Smith, and A. Aderem. 1999. Toll-like receptor-2 mediates mycobacteria-induced proinflammatory signaling in macrophages. Proc Natl Acad Sci U S A 96:14459-14463.
19.Kawai, T., O. Adachi, T. Ogawa, K. Takeda, and S. Akira. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11:115-122.
20.Horng, T., G. M. Barton, R. A. Flavell, and R. Medzhitov. 2002. The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420:329-333.
21.Kyriakis, J. M., and J. Avruch. 2001. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807-869.
22.Wang, C., L. Deng, M. Hong, G. R. Akkaraju, J. Inoue, and Z. J. Chen. 2001. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412:346-351.
23.Chang, L., and M. Karin. 2001. Mammalian MAP kinase signalling cascades. Nature 410:37-40.
24.Sancho, R., A. S. Nateri, A. G. de Vinuesa, C. Aguilera, E. Nye, B. Spencer-Dene, and A. Behrens. 2009. JNK signalling modulates intestinal homeostasis and tumourigenesis in mice. EMBO J 28:1843-1854.
25.Treinies, I., H. F. Paterson, S. Hooper, R. Wilson, and C. J. Marshall. 1999. Activated MEK stimulates expression of AP-1 components independently of phosphatidylinositol 3-kinase (PI3-kinase) but requires a PI3-kinase signal To stimulate DNA synthesis. Mol Cell Biol 19:321-329.
26.Chen, Z. J. 2005. Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol 7:758-765.
27.Deng, L., C. Wang, E. Spencer, L. Yang, A. Braun, J. You, C. Slaughter, C. Pickart, and Z. J. Chen. 2000. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103:351-361.
28.Jenkins, K. A., and A. Mansell. 2010. TIR-containing adaptors in Toll-like receptor signalling. Cytokine 49:237-244.
29.Ghosh, S., and M. Karin. 2002. Missing pieces in the NF-kappaB puzzle. Cell 109 Suppl:S81-96.
30.Hansson, G. K. 2005. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 352:1685-1695.
31.Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu Rev Immunol 20:197-216.
32.Simionescu, M. 2007. Implications of early structural-functional changes in the endothelium for vascular disease. Arterioscler Thromb Vasc Biol 27:266-274.
33.Jonasson, L., J. Holm, O. Skalli, G. Gabbiani, and G. K. Hansson. 1985. Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis. J Clin Invest 76:125-131.
34.Yan, Z. Q., and G. K. Hansson. 2007. Innate immunity, macrophage activation, and atherosclerosis. Immunol Rev 219:187-203.
35.1998. Inflammatory and Immune Mechanisms in Vascular Disease. Bethesda, Maryland, USA. February 19-20, 1998. Abstracts. J Vasc Surg 27:1152-1192.
36.Mullick, A. E., P. S. Tobias, and L. K. Curtiss. 2005. Modulation of atherosclerosis in mice by Toll-like receptor 2. J Clin Invest 115:3149-3156.
37.Kazemi, M. R., C. M. McDonald, J. K. Shigenaga, C. Grunfeld, and K. R. Feingold. 2005. Adipocyte fatty acid-binding protein expression and lipid accumulation are increased during activation of murine macrophages by toll-like receptor agonists. Arterioscler Thromb Vasc Biol 25:1220-1224.
38.Makowski, L., J. B. Boord, K. Maeda, V. R. Babaev, K. T. Uysal, M. A. Morgan, R. A. Parker, J. Suttles, S. Fazio, G. S. Hotamisligil, and M. F. Linton. 2001. Lack of macrophage fatty-acid-binding protein aP2 protects mice deficient in apolipoprotein E against atherosclerosis. Nat Med 7:699-705.
39.Davies, M. J., J. L. Gordon, A. J. Gearing, R. Pigott, N. Woolf, D. Katz, and A. Kyriakopoulos. 1993. The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol 171:223-229.
40.Liu, X., T. Ukai, H. Yumoto, M. Davey, S. Goswami, F. C. Gibson, 3rd, and C. A. Genco. 2008. Toll-like receptor 2 plays a critical role in the progression of atherosclerosis that is independent of dietary lipids. Atherosclerosis 196:146-154.
41.Nilsson, L. M., Z. W. Sun, J. Nilsson, I. Nordstrom, Y. W. Chen, J. D. Molkentin, D. Wide-Swensson, P. Hellstrand, M. L. Lydrup, and M. F. Gomez. 2007. Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation. Am J Physiol Cell Physiol 292:C1167-1178.
42.Grassl, C., B. Luckow, D. Schlondorff, and U. Dendorfer. 1999. Transcriptional regulation of the interleukin-6 gene in mesangial cells. J Am Soc Nephrol 10:1466-1477.
43.Wang, D., Z. Liu, Q. Li, M. Karpurapu, V. Kundumani-Sridharan, H. Cao, N. Dronadula, F. Rizvi, A. K. Bajpai, C. Zhang, G. Muller-Newen, K. W. Harris, and G. N. Rao. 2007. An essential role for gp130 in neointima formation following arterial injury. Circ Res 100:807-816.
44.Schindler, U., and V. R. Baichwal. 1994. Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol Cell Biol 14:5820-5831.
45.Ohbayashi, N., O. Ikeda, N. Taira, Y. Yamamoto, R. Muromoto, Y. Sekine, K. Sugiyama, T. Honjoh, and T. Matsuda. 2007. LIF- and IL-6-induced acetylation of STAT3 at Lys-685 through PI3K/Akt activation. Biol Pharm Bull 30:1860-1864.
46.Martin, M., K. Rehani, R. S. Jope, and S. M. Michalek. 2005. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 6:777-784.
47.Martin, M., R. E. Schifferle, N. Cuesta, S. N. Vogel, J. Katz, and S. M. Michalek. 2003. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide. J Immunol 171:717-725.
48.Salaun, B., P. Romero, and S. Lebecque. 2007. Toll-like receptors' two-edged sword: when immunity meets apoptosis. Eur J Immunol 37:3311-3318.
49.Kavurma, M. M., and L. M. Khachigian. 2003. ERK, JNK, and p38 MAP kinases differentially regulate proliferation and migration of phenotypically distinct smooth muscle cell subtypes. J Cell Biochem 89:289-300.
50.Bhoumik, A., and Z. Ronai. 2008. ATF2: a transcription factor that elicits oncogenic or tumor suppressor activities. Cell Cycle 7:2341-2345.
51.Salameh, A., F. Galvagni, F. Anselmi, C. De Clemente, M. Orlandini, and S. Oliviero. 2010. Growth factor stimulation induces cell survival by c-Jun. ATF2-dependent activation of Bcl-XL. J Biol Chem 285:23096-23104.
52.Hai, T., and M. G. Hartman. 2001. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 273:1-11.
53.Ma, Q., X. Li, D. Vale-Cruz, M. L. Brown, F. Beier, and P. LuValle. 2007. Activating transcription factor 2 controls Bcl-2 promoter activity in growth plate chondrocytes. J Cell Biochem 101:477-487.
54.Medzhitov, R., and T. Horng. 2009. Transcriptional control of the inflammatory response. Nat Rev Immunol 9:692-703.
55.Ghosh, S., and M. S. Hayden. 2008. New regulators of NF-kappaB in inflammation. Nat Rev Immunol 8:837-848.
56.Ollivier, V., G. C. Parry, R. R. Cobb, D. de Prost, and N. Mackman. 1996. Elevated cyclic AMP inhibits NF-kappaB-mediated transcription in human monocytic cells and endothelial cells. J Biol Chem 271:20828-20835.
57.Parry, G. C., and N. Mackman. 1997. Role of cyclic AMP response element-binding protein in cyclic AMP inhibition of NF-kappaB-mediated transcription. J Immunol 159:5450-5456.
58.Burleigh, M. E., V. R. Babaev, P. G. Yancey, A. S. Major, J. L. McCaleb, J. A. Oates, J. D. Morrow, S. Fazio, and M. F. Linton. 2005. Cyclooxygenase-2 promotes early atherosclerotic lesion formation in ApoE-deficient and C57BL/6 mice. J Mol Cell Cardiol 39:443-452.
59.Fukata, M., A. Chen, A. Klepper, S. Krishnareddy, A. S. Vamadevan, L. S. Thomas, R. Xu, H. Inoue, M. Arditi, A. J. Dannenberg, and M. T. Abreu. 2006. Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: Role in proliferation and apoptosis in the intestine. Gastroenterology 131:862-877.
60.Krishnan, J., K. Selvarajoo, M. Tsuchiya, G. Lee, and S. Choi. 2007. Toll-like receptor signal transduction. Exp Mol Med 39:421-438.
61.Cole, J. E., E. Georgiou, and C. Monaco. 2010. The expression and functions of toll-like receptors in atherosclerosis. Mediators Inflamm 2010:393946.
62.Huang, G., L. Z. Shi, and H. Chi. 2009. Regulation of JNK and p38 MAPK in the immune system: signal integration, propagation and termination. Cytokine 48:161-169.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top