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系統識別號 U0026-0812200912003627
論文名稱(中文) 人類凝血酶調節素之類凝集素功能區於敗血症及粥狀動脈硬化抗發炎作用之研究
論文名稱(英文) The Anti-inflammatory Functions of Thrombomodulin Lectin-like Domain in Sepsis and Atherosclerosis
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 94
學期 2
出版年 95
研究生(中文) 郭冠麟
研究生(英文) Kuan-Lin Kuo
電子信箱 whiterailway@yahoo.com.tw
學號 t3693403
學位類別 碩士
語文別 英文
論文頁數 76頁
口試委員 指導教授-吳華林
口試委員-施桂月
口試委員-林淑華
口試委員-林尊湄
中文關鍵字 凝血酶調節素  發炎反應  粥狀動脈硬化  敗血症 
英文關鍵字 Inflammation  Thrombomodulin  Sepsis  Atherosclerosis 
學科別分類
中文摘要 凝血酶調節素(Thrombomodulin, TM)為一種表現在血管內皮細胞表面的醣化蛋白質,可做為凝血酶(thrombin)的接受器。當凝血酶與凝血酶調節素結合後,會抑制凝血酶促凝血的活性,轉而活化蛋白質C(protein C),進一步達到抗凝血功能。近期的研究文獻發表指出,凝血酶調節素除了抗凝血功能以外,在其他多種生理功能中扮演重要的角色。我們研究發現凝血酶調節素之N端類凝集素功能區(N-terminal lectin-like domain, TMD1)具有獨特的抗發炎效能。在此研究中主要探討:在酯多醣(lipopolysaccharide, LPS)及克雷白氏肺炎桿菌(Klebsiella pneumonia, K. pneumoniae)所引發的敗血性急性發炎反應,以及在慢性發炎疾病粥狀動脈硬化(atherosclerosis)病變過程中,TMD1對於此急性及慢性發炎反應之抑制功效。我們利用酵母菌表現系統,表現TMD1重組蛋白質,進而經由親和性鎳離子螯合樹脂管柱,純化出高純度的TMD1重組蛋白質。在急性發炎反應的細胞實驗中,我們證實, TMD1能有效抑制LPS所誘發之巨噬細胞產生的發炎因子第六介白素(IL-6)、腫瘤壞死因子α(TNF-α),並且能經由抑制誘導型一氧化氮合成酶(iNOS)表現量而降低一氧化氮(NO)的生成。在LPS誘發之發炎反應訊息傳遞路徑中,TMD1會降低LPS所誘發的ERK1/2及p38磷酸化。同樣地,TMD1亦會抑制LPS誘發之IκBα磷酸化及水解,進一步避免NF-κB轉位由細胞質進入細胞核中,最終能使得下游的促發炎因子減少,有效減輕發炎的程度。在敗血症研究的動物模式中,將TMD1以尾靜脈注射方式打入老鼠體內,並以腹腔注射注入LPS以及K. pneumoniae菌液,用以誘發急性發炎敗血症。實驗結果發現,TMD1能顯著抑制LPS以及K. pneumoniae引起之血清TNF-α與NO生成量。而在病理組織切片中,亦可以觀察到TMD1能有效抑制LPS以及K. pneumoniae引起的肺臟、腎臟之發炎病變以及減緩敗血症引起之致死率。在TMD1的轉殖鼠當中,打入致死劑量的K. pneumoniae菌液,也發現TMD1轉殖鼠死亡率遠比同源正常鼠來的低。另一方面,我們也探討了TMD1在慢性發炎中扮演的角色。在細胞實驗中,證實TMD1會干擾單核球附著在活化的人類內皮細胞上,並且能進一步抑制單核球transmigration穿越內皮細胞層,因此,TMD1可能經由抑制單核球轉移至內皮細胞層下,而達到抑制粥狀動脈硬化的能力。除了敗血症急性發炎的研究以外,我們利用載體蛋白E基因剔除老鼠(Apo E-/- mice)作為慢性發炎的動物實驗模式,此實驗證實TMD1可以抑制Apo E-/- 老鼠主動脈上粥狀硬化動脈斑的生成,降低了粥狀動脈硬化的程度。此外,在Apo E-/- 老鼠的心臟組織萃取液中,TMD1能降低心血管系統中黏著分子VCAM-1以及ICAM-1的表現量,反之,卻能避免細胞的間質蛋白cadherin之表現降低。TMD1能經由抑制慢性發炎反應所誘發之黏著分子表現增加及避免間質蛋白表現的減少,進而減緩動脈粥狀硬化的形成。從以上結果我們推論,TM在急性及慢性發炎反應中扮演重要的角色,且暗示著TMD1可在敗血症及粥狀動脈硬化的治療上,提供新的思考方向。
英文摘要 Thrombomodulin (TM), a thrombin receptor on endothelial cell membrane, inhibits the procoagulant functions of thrombin and acts as a protein cofactor in thrombin-catalyzed activation of protein C. Recent studies indicate that TM not only regulates coagulation but also plays an important role in other physiological functions. Our group has found that the TM N-terminal lectin-like domain (TMD1) has unique anti-inflammatory properties. In this study, we explored the effect of TMD1 on lipopolysaccharide (LPS) and Klebsiella pneumoniae (K. pneumoniae)-induced acute inflammation in sepsis, and the effect of TMD1 on atherosclerosis as a chronic inflammation model. The DNA fragment of human TMD1 was cloned into the plasmid for Pichia pastoris protein expression system. The yeast-expressed TMD1 protein was purified with a nickel-chelated Sepharose chromatography and further identified with specific antibody. In the in vitro study, we found that TMD1 could suppress LPS-induced secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), and synthesis of nitric oxide (NO) via inhibition of inducible nitric oxide synthase (iNOS) expression in Raw 264.7 macrophages. In the study of the mitogen activated protein kinase (MAPK) signaling pathway, we found that TMD1 could suppress LPS-induced extracellular signal-regulated kinase1/2 (ERK1/2) and p38 phosphorylation. Also, TMD1 inhibited LPS-induced nuclear translocation of p65 and p50 from cytosol to nuclear via modulating phosphorylation and degradation of jnhibitor of nuclear factor-kappa B alpha (IκBα) in the nuclear factor-kappa B (NF-κB) signaling pathway. In the study of sepsis in vivo, TMD1 was administrated intravenously into mice 30 min before infusion with LPS and K. pneumoniae. The results showed that TMD1 attenuated LPS and K. pneumoniae-induced serum TNF-α and NO elevation, lung and renal injuries, and mortality on mice. Moreover, in exposure TMD1 transgenic mice to lethal K. pneumoniae, TMD1 transgenic mice exhibited reduced mortality in comparison with littermate normal mice. In addition, we study the effect of TMD1 on chronic inflammatory disease as in atherosclerosis using in vivo and in vitro models. In the in vitro study, we also found that TMD1 could inhibit adhesion of monocytes to endothelium as well as their transmigration through the endothelium. In ApoE knockout (ApoE-/-) mice, we elucidated that TMD1 could reduce plaque formations in the aortas of ApoE-/- mice. Therefore, TMD1 could inhibit VCAM-1 and ICAM-1 expression and prevent cadherin down-regulation in cardiovascular system of ApoE-/- mice. In conclusion, we demonstrated that TMD1 has novel anti-inflammatory activities through suppressing not only LPS and K. pneumoniae-induced acute inflammation via MAPK and NF-κB signaling pathway, but also chronic inflammation among atherosclerosis via inhibiting interaction between leukocyte and endothelial cells. Therefore, TMD1 may be a novel therapeutic agent in treatment of different inflammatory pathological situations including sepsis and atherosclerosis.
論文目次 I Abstract 1
II Chinese Abstract 2
III Acknowledgments 3
IV Content Table 4
V Introduction 8
VI Specific Aims 13
VII Materials and Methods 14
VIII Results 29
IX Discussion 34
X References 38
XI Figures and Legends 42
XII Tables 59
XIII Reagents, Drugs and Chemicals 60
XIV Instruments 65
XV Abbreviations 67
XVI Appendixes 69
XVII Resume 76
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