臺灣博碩士論文加值系統

動脈粥狀硬化 (atherosclerosis) 被認為是血管系統慢性發炎的疾病。單核球 (monocytes) 的橫越內皮移動 (transendothelial migration) 並分化為單核球衍生巨噬細胞 (monocyte-derived macrophages) 為動脈粥狀硬化過程中的關鍵要素之一。單核球衍生巨噬細胞釋放促發炎因子 (proinflammatory factors) 使內皮細胞增加沾粘蛋白 (adhesion molecules) ICAM-1及VCAM-1 之基因表現。本研究以phorbol 12-myristate 13-acetate (PMA) 刺激單核球細胞株U937，使其分化成巨噬細胞，收集巨噬細胞之條件培養液 (conditioned medium)， 並利用此條件培養液刺激人類臍靜脈內皮細胞 (human umbilical vein endothelial cells)，觀察內皮細胞之ICAM-1及VCAM-1基因表現。結果發現，條件培養液分別經由ERK/AP-1以及Akt/NF-kB之訊息傳遞路徑使 ICAM-1 及 VCAM-1 之基因表現增加。本研究提供了巨噬細胞刺激內皮細胞表現ICAM-1及VCAM-1之分子調控機轉。

Atherosclerosis is a chronic inflammatory disease of the vascular system. Transmigration of monocytes during inflammation is key elements in atherosclerosis. Monocyte-derived macrophages release proinflammatory factors which are responsible for the upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells.
In the present study, Incubation with conditioned medium derived from phorbol 12-myristate 13-acetate (PMA)-stimulated U937 macrophages induced ICAM-1 and VCAM-1 mRNA expression in human umbilical vein endothelial cells (HUVECs). Conditioned medium also increased ERK and Akt phosphorylation. In addition, macrophage-induced ICAM-1 and VCAM-1 expression was mediated by ERK/AP-1 signaling pathway and Akt/NF-kB signaling pathway, respectively. Our findings provide insights into the mechanisms underlying the macrophage induction of ICAM-1 and VCAM-1 expression in ECs.

摘要 1
ABSTRACT 2
第一章、前言 5
第一節、動脈硬化簡介 5
第二節、動脈硬化成因 5
內皮細胞功能失調 5
脂肪條塊生成 7
深入性病灶 7
不穩定性之斑塊 8
第三節、動脈硬化中白血球與血管壁之互動關係 8
第四節、內皮細胞與HUVEC之簡介 9
第五節、U937 CELL LINE之特性介紹 11
第六節、MACROPHAGE 的種類及功能 12
第七節、動脈硬化與黏附因子ICAM-1與VCAM-1之簡介 13
類免疫球蛋白分子 13
VCAM-1 14
ICAM-1 14
第八節、NF-kB、AP-1之介紹 15
NF-kB 15
AP-1 16
第二章、材料與方法 17
第一節、材料 17
第二節、方法 19
初級臍帶靜脈內皮細胞培養 19
臍帶靜脈內皮細胞繼代培養 19
西方點墨法 20
Conditioned Medium 之製備 21
Conditioned medium 之使用 21
RT-PCR之primer設計 22
Real-time PCR 22
第三章、實驗結果 24
第一節、以CONDITION MEDIUM培養內皮細胞之時間點及CONDITION MEDIUM之濃度差異 24
第二節、以CONDITION MEDIUM加以CYTOKINE抑制劑測試之結果 25
第三節、利用西方點墨法 (WESTERN–BLOTTING)觀察蛋白磷酸化之情況 25
第四節、利用抑制劑證實經CM作用後內皮細胞下游之訊息傳遞路徑 26
第五節、利用REAL-TIME PCR技術再一次驗證前面之結果 27
第六節、訊息傳遞路徑 27
第四章、討論 29
第五章、參考文獻 32
第六章、圖附錄 39

1.The pathogenesis of atherosclerosis: a perspective for the 1990s. Ross R (1993) Nature, 362, 801-809.
2.The pathogenesis of atherosclerosis. Hegele RA (1996) Clin Chim Acta, 246, 21-38.
3.Adhesion molecules--Part 1. Frenette PS and Wagner DD (1996) New England Journal of Medicine, 334, 1526-1529.
4.Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation. Huh HY, Pearce SF, Yesner LM, Schindler JL, and Silverstein RL (1996) Barbaux S Blood, 87, 2020-2028.
5.Cellular adhesion molecules and atherogenesis. American Journal of Medicine, Price DT and Loscalzo J (1999). 107, 85-97.
6.Adhesion molecules and atherosclerosis. Blankenberg S, Barbaux S, and Tiret L (2003).Atherosclerosis, 170, 191-203.
7.Interleukin-1beta-induced cyclooxygenase-2 expression requires activation of both c-Jun NH2-terminal kinase and p38 MAPK signal pathways in rat renal mesangial cells. Guan Z, Buckman SY, Miller BW, Springer LD, and Morrison AR (1998). Journal of Biological Chemistry, 273, 28670-28676.
8.Cellular adhesion molecules on vascular smooth muscle cells. Cardiovascular Research, Braun M, Pietsch P, Schror K, Baumann G, and Felix SB (1999). 41, 395-401.
9.Interleukin-1 gene expression in rabbit vascular tissue in vivo. Clinton SK, Fleet JC, Loppnow H, Salomon RN, Clark BD, Cannon JG, Shaw AR, Dinarello CA, and Libby P (1991). American Journal of Pathology, 138.
10.Oxidized low density lipoprotein induces differentiation adhesion of human monocytes the monocytic cell line U937. Frostegard J, Nilsson J, Haegerstr A, Hamsten A, Wigzell H, Gidlund M. (1990). Proc Natl Acad Sci USA 87(3): 904-908.
11.The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Oleinick NL, Morris RL, and Belichenko I (2002). Photochemical & Photobiological Sciences, 1, 1-21.
12.Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury. Thyberg J (1998). Histology & Histopathology, 13, 871-891.
13.Signal transduction through MAP kinase cascades. Lewis TS, Shapiro PS, and Ahn NG (1998). Advances in Cancer Research, 74, 49-139.
14.LDLs induce fibroblast spreading independently of the LDL receptor via activation of the p38 MAPK pathway. Dobreva I, Waeber G, Mooser V, James RW, Widmann C Ito H (2003).Journal of lipid Research, 44, 2382-90.
15.Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Kyriakis JM and Avruch J (2001). Physiological Reviews, 81, 807-869.
16.Epstein-Barr virus-encoded dUTPase enhances proinflammatory cytokine production by macrophages in contact with endothelial cells: evidence for depression-induced atherosclerotic risk. Waldman WJ, Williams MV Jr, Lemeshow S, Binkley P, Guttridge D, Kiecolt-Glaser JK, Knight DA, Ladner KJ, Glaser R. Brain Behav Immun. 2008 February ; 22(2): 215–223.
17.Epstein-Barr Virus (EBV)-Infected Monocytes Facilitate Dissemination of EBV within the Oral Mucosal Epithelium. Tugizov S, Herrera R, Veluppillai P, Greenspan J, Greenspan D, Palefsky JM. Journal of Virology 2007;81:5484–5496.
18.EBV-encoded dUTPase induces immune dysregulation: Implications for the pathophysiology of EBV-associated disease. Glaser R, Litsky ML, Padgett DA, Baiocchi RA, Yang EV, Chen M, Yeh PE, Green-Church KB, Caligiuri MA, Williams MV. Virology 2006;346:205–218.
19.Cornuside suppresses cytokine-induced proinflammatory and adhesion molecules in the human umbilical vein endothelial cells. Kang DG, Moon MK, Lee AS, Kwon TO, Kim JS, Lee HS. Biol Pharm Bull. 2007 Sep;30(9):1796-9.
20.Epstein Barr virus specific T-cells generated from unstable human atherosclerotic lesions: Implications for plaque inflammation. de Boer OJ, Teeling P, Idu MM, Becker AE, van der Wal AC. Atherosclerosis 2006;184:322–329
21.Tripterine inhibits the expression of adhesion molecules in activated endothelial cells. Zhang DH, Marconi A, Xu LM, Yang CX, Sun GW, Feng XL, Ling CQ, Qin WZ, Uzan G, d'Alessio P. J Leukoc Biol. 2006 Aug;80(2):309-19. Epub 2006 Jun 12.
22.Bile acids induce adhesion molecule expression in endothelial cells through activation of reactive oxygen species, NF-kappaB, and p38. Qin P, Tang X, Elloso MM, Harnish DC. Am J Physiol Heart Circ Physiol. 2006 Aug;291(2):H741-7. Epub 2006 Mar 31.
23.The isoprostane 8-iso-PGF2alpha suppresses monocyte adhesion to human microvascular endothelial cells via two independent mechanisms. Kumar A, Kingdon E, Norman J. FASEB J. 2005 Mar;19(3):443-5. Epub 2005 Jan 7.
24.Respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression. Arnold R, K�圢ig W. J Immunol. 2005 Jun 1;174(11):7359-67.
25.Flavones mitigate tumor necrosis factor-alpha-induced adhesion molecule upregulation in cultured human endothelial cells: role of nuclear factor-kappa B. Choi JS, Choi YJ, Park SH, Kang JS, Kang YH.J Nutr. 2004 May;134(5):1013-9.
26.Atomic force microscopy measurement of leukocyte-endothelial interaction. Zhang X, Chen A, De Leon D, Li H, Noiri E, Moy VT, Goligorsky MS. Am J Physiol Heart Circ Physiol. 2004 Jan;286(1):H359-67. Epub 2003 Sep 11.
27.P-cresol, a uremic toxin, decreases endothelial cell response to inflammatory cytokines. Dou L, Cerini C, Brunet P, Guilianelli C, Moal V, Grau G, De Smet R, Vanholder R, Sampol J, Berland Y. Kidney Int. 2002 Dec;62(6):1999-2009.
28.Persistent infection of human microvascular endothelial cells by coxsackie B viruses induces increased expression of adhesion molecules. Zanone MM, Favaro E, Conaldi PG, Greening J, Bottelli A, Perin PC, Klein NJ, Peakman M, Camussi G. J Immunol. 2003 Jul 1;171(1):438-46.
29.Omega-3 fatty acids suppress monocyte adhesion to human endothelial cells: role of endothelial PAF generation.Mayer K, Merfels M, Muhly-Reinholz M, Gokorsch S, Rosseau S, Lohmeyer J, Schwarzer N, Kr�刜l M, Suttorp N, Grimminger F, Seeger W. Am J Physiol Heart Circ Physiol. 2002 Aug;283(2):H811-8.
30.Probucol inhibits oxidized-low density lipoprotein-induced adhesion of monocytes to endothelial cells in vitro. Liu GX, Ou DM, Li LX, Chen LX, Huang HL, Liao DF, Tang CS. Acta Pharmacol Sin. 2002 Jun;23(6):516-22.
31.Fimbria-dependent activation of cell adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells. Khlgatian M, Nassar H, Chou HH, Gibson FC 3rd, Genco CA. Infect Immun. 2002 Jan;70(1):257-67.
32.Role of phosphoinositide 3-kinase in monocyte recruitment under flow conditions. Gerszten RE, Friedrich EB, Matsui T, Hung RR, Li L, Force T, Rosenzweig A. J Biol Chem. 2001 Jul 20;276(29):26846-51. Epub 2001 Mar 1.
33.Interaction between chronically HIV-infected promonocytic cells and human umbilical vein endothelial cells: role of proinflammatory cytokines and chemokines in viral expression modulation. Borghi MO, Panzeri P, Shattock R, Sozzani S, Dobrina A, Meroni PL. Clin Exp Immunol. 2000 Apr;120(1):93-100.
34.Orins and lipopolysaccharide (LPS) from Salmonella typhimurium induce leucocyte transmigration through human endothelial cells in vitro. Galdiero M, Folgore A, Molitierno M, Greco R. Clin Exp Immunol. 1999 Jun;116(3):453-61.
35.Factor Xa induces cytokine production and expression of adhesion molecules by human umbilical vein endothelial cells. Senden NH, Jeunhomme TM, Heemskerk JW, Wagenvoord R, van't Veer C, Hemker HC, Buurman WA. J Immunol. 1998 Oct 15;161(8):4318-24.
36.Modulation of monocyte-endothelial cell interactions by platelet microparticles. Barry OP, Pratic�� D, Savani RC, FitzGerald GA. J Clin Invest. 1998 Jul 1;102(1):136-44.
37.Cytokine and adhesion molecule expression in human monocytes and endothelial cells stimulated with bacterial heat shock proteins. Galdiero M, de l'Ero GC, Marcatili A. Infect Immun. 1997 Feb;65(2):699-707.
38.IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes. Watson C, Whittaker S, Smith N, Vora AJ, Dumonde DC, Brown KA. Clin Exp Immunol. 1996 Jul;105(1):112-9.
39.Intercellular adhesion molecule-1 mediates the expression of monocyte-derived MIP-1 alpha during monocyte-endothelial cell interactions. Lukacs NW, Strieter RM, Elner VM, Evanoff HL, Burdick M, Kunkel SL. Blood. 1994 Mar 1;83(5):1174-8.
40.Expression of vascular cell adhesion molecule-1 in fibroblastlike synoviocytes after stimulation with tumor necrosis factor. Marlor CW, Webb DL, Bombara MP, Greve JM, Blue ML. Am J Pathol. 1992 May;140(5):1055-60.
41.Prolonged exposure to high insulin impairs the endothelial PI3-kinase/Akt/nitric oxide signalling. Madonna R, De Caterina R. Thromb Haemost. 2009 Feb;101(2):345-50.
42.Effect of Buddleja officinalis on high-glucose-induced vascular inflammation in human umbilical vein endothelial cells. Lee YJ, Kang DG, Kim JS, Lee HS. Exp Biol Med (Maywood). 2008 Jun;233(6):694-700. Epub 2008 Apr 11.
43.PM2.5 and PM10 induce the expression of adhesion molecules and the adhesion of monocytic cells to human umbilical vein endothelial cells. Montiel-D�償alos A, Alfaro-Moreno E, L�櫝ez-Marure R. Inhal Toxicol. 2007;19 Suppl 1:91-8.
44.Coenzyme Q10 prevents high glucose-induced oxidative stress in human umbilical vein endothelial cells. Tsuneki H, Sekizaki N, Suzuki T, Kobayashi S, Wada T, Okamoto T, Kimura I, Sasaoka T. Eur J Pharmacol. 2007 Jul 2;566(1-3):1-10. Epub 2007 Mar 19.
45.Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells. Pandolfi A, Di Pietro N, Sirolli V, Giardinelli A, Di Silvestre S, Amoroso L, Di Tomo P, Capani F, Consoli A, Bonomini M. J Cell Physiol. 2007 Dec;213(3):699-709.
46.Respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression. Arnold R, K�圢ig W. J Immunol. 2005 Jun 1;174(11):7359-67.