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系統識別號 U0026-0812200915221088
論文名稱(中文) 凝血酶調節素類表皮生長因子與富含絲胺酸/息寧胺酸功能區藉由結合凝血酶抑制動脈粥狀硬化
論文名稱(英文) EGF-like and serine/threonine-rich domains of thrombomodulin suppress atherosclerosis by binding thrombin
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 97
學期 2
出版年 98
研究生(中文) 魏希儒
研究生(英文) Hsi-Ju Wei
電子信箱 T3696408@mail.ncku.edu.tw
學號 t3696408
學位類別 碩士
語文別 英文
論文頁數 85頁
口試委員 口試委員-林淑華
指導教授-吳華林
口試委員-施桂月
口試委員-張文粲
中文關鍵字 凝血酶  動脈粥狀硬化  凝血酶調節素 
英文關鍵字 Thrombin  Atherosclerosis  Thrombomodulin 
學科別分類
中文摘要 動脈粥狀硬化 (atherosclerosis) 為慢性發炎疾病,可被分為早期與晚期兩種病變進程。凝血酶 (thrombin, TH) 藉由活化蛋白酶激活受體-1 (protease-activated receptor-1, PAR-1) 以調控動脈粥狀硬化病變的組成和動脈粥狀硬化的形成過程。凝血酶調節素 (thrombomodulin, TM) 為細胞表面的穿膜醣蛋白,主要生理功能是結合 TH 達到抗凝血作用,其蛋白結構從胺基端依序被分為 lectin-like domain (TMD1)、六個 EGF-like domain (TMD2)、serine/threonine-rich region (TMD3)、transmembrane domain (D4) 及碳端的cytoplasmic tail (D5)。之前的研究中發現,重組的 TMD123 能有效降低小鼠頸動脈綁結手術模式中的新生內膜形成 (neointima formation),此外 TMD1 具有直接抑制發炎反應的功能,但是對於 TMD23 與 TH 對動脈粥狀硬化之作用及其調控機制仍尚未明瞭。本研究利用酵母菌表現系統製備小鼠重組 TMD23 野生型及三個 TMD23 突變蛋白,分別突變於與 TH 結合相關位置的I441A、D440A/D442A 及 D440A/I441A/D442A。TMD23 野生型及 TMD23 (D440A/D442A) 能夠有效結合 TH 、活化蛋白 C 、及延長 TH 引起的凝血時間,然而 TMD23 (I441A) 及 TMD23 (D440A/I441A/D442A) 皆失去這些功能。在人類臍靜脈內皮細胞 (human umbilical vein endothelial cells, HUVECs)上,我們發現TMD23 野生型可減低 TH 活化細胞膜上 PAR-1 之後的內吞作用及TH誘導的細胞質鈣離子濃度上升,但 TMD23 (I441A) 則無此作用。此外,TMD23 野生型及 TMD23 (D440A/D442A) 能有效降低 TH 在 HUVECs 中所引起的通透性增加現象,及抑制 TH 誘導細胞間黏附分子-1 (intercellular adhesion molecule, ICAM-1)、血管細胞黏附分子-1 (vascular cell adhesion molecule, VCAM-1)、單核細胞趨化蛋白-1 (monocyte chemoattractant protein-1, MCP-1) 的表現,然而 TMD23 (I441A) 及 TMD23 (D440A/I441A/D442A) 則沒有影響。在小鼠巨噬細胞 (RAW264.7 cells), TMD23 野生型及 TMD23 (D440A/D442A) 能有效降低 TH 誘導的腫瘤壞死因子α (TNFα)表現,但 TMD23 (I441A) 及 TMD23 (D440A/I441A/D442A) 則沒有影響。此外 TMD23 及三種突變蛋白皆無法抑制 TH 引起的平滑肌細胞 (A7r5 cells) 增殖作用。進一步在小鼠頸動脈綁結手術模式中,TMD2 (EGF1-3)、TMD2 (EGF4-6)、TMD23 野生型及三種TMD23 突變蛋白皆能有效抑制新生內膜形成,且其中 TMD2 (EGF4-6)、TMD23 野生型及 TMD23 (D440A/D442A) 之抑制效果更為顯著。另外在載體蛋白E基因剔除老鼠(ApoE -/- mice)中,TMD2 (EGF4-6)、TMD23 野生型及 TMD23 (D440A/D442A)能有效降低主動脈斑塊產生。綜合以上結果,TMD23 可藉由結合 TH 對 動脈粥狀硬化的形成有抑制的效果。
英文摘要 Atherosclerosis, a chronic inflammatory disease, can be divided into early lesion and advanced lesion. Thrombin (TH) modulates the formation of the atherosclerotic lesion and the course of atherogenesis by stimulating a variety of cellular effects through protease-activated receptor-1 (PAR-1). Thrombomodulin (TM) is a membrane-bound glycoprotein and contains a N-terminal lectin-like domain (D1), six epidermal growth factor (EGF)-like repeats (D2), a serine/threonine-rich region (D3), a transmembrane domain (D4) and a short cytoplasmic tail (D5). The main physiological function of TM is to bind thrombin. In our previous study, we found that recombinant TM containing all extracellular domains of TM (TMD123) significantly decreased neointima formation in mouse carotid ligation model. We also demonstrated that TM lectin like domain (TM domain 1,TMD1) has direct anti-inflammatory effect. However, the roles of TM domains 2 & 3 (TMD23) and thrombin in atherosclerosis are still unclear. The mouse recombinant TMD23-wild type and three TMD23 mutants at thrombin binding site, I441A, D440A/D442A, and D440A/I441A/D442A were constructed and purified. TMD23-wild type and TMD23 (D440A/D442A) could effectively bind to thrombin, activate protein C and prolong the thrombin clotting time while TMD23 (I441A) and TMD23 (D440A/I441A/D442A) lost these functions. We found that TMD23-wild type, but not TMD23 (I441A), could decrease the thrombin-induced internalization of the surface PAR-1 and decrease the thrombin-induced cytoplasmic Ca2+ release in human umbilical vein endothelial cells (HUVECs). TMD23-wild type and TMD23 (D440A/D442A) could significantly decrease thrombin-induced intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and permeability in HUVECs; however, TMD23 (I441A) and TMD23 (D440A/I441A/D442A) had no effect. We also found that TMD23-wild type and TMD23 (D440A/D442A), but not TMD23 (I441A) and TMD23 (D440A/I441A/D442A) could significantly decrease thrombin-induced tumor necrosis factor α (TNFα) in RAW 264.7 cells. However, all of TMD23-wild type and mutants had no effects on the proliferation of smooth muscle cells (A7r5 cells). Furthermore, We found that TMD2 (EGF1-3), TMD2 (EGF4-6), TMD23-wild type, TMD23 (I441A), TMD23 (D440A/D442A), and TMD23 (D440A/I441A/D442A) could significantly reduce carotid neointima formation in the C57BL/6 mice. However, TMD2 (EGF4-6), TMD23-wild type, and TMD23 (D440A/D442A) were more effective than TMD2 (EGF1-3), TMD23 (I441A), and TMD23 (D440A/I441A/D442A) in reducing carotid neointima formation. In addition, TMD2 (EGF4-6), TMD23-wild type, and TMD23 (D440A/D442A) significantly decreased the severity of atherosclerosis in the ApoE -/- mice. In conclusion, TMD23 could significantly reduce atherosclerosis and neointima formation in mice through binding thrombin.
論文目次 Section Content Page
I. Chinese Abstract 2
II. Abstract 3
III. Acknowledgment 4-5
IV. Content Table 6-8
V. Abbreviation 9-10
VI Instruments 11-12
VII Reagents 13-16
VIII. Introduction 17-22
A Atherosclerosis 17
B Thrombin 17
C Thrombin in atherosclerosis 18
D Thrombomodulin 18
E Animal models of experimental atherosclerosis 20
F Gene targeting atherosclerotic mouse models 20
G Carotid intima-media thickening mouse model 21
IX. Specific Aims 23
X. Materials and Methods 24-48
A Quick-change site-directed mutagenesis 24
B Expression and purification of recombinant mTMD23-wild type and three mutant proteins 27
C Thrombin solid phase assay 33
D Thrombin dependent protein C activation assay 34
E Thrombin clotting time assay 35
F Cell culture 36
G Effects of TMD23-wild type or TMD23 (I441A) treatment on thrombin-induced internalization of the surface PAR-1 in HUVECs. 38
H Effects of TMD23-wild type or TMD23 (I441A) treatment on thrombin-induced cytoplasmic Ca2+ release in HUVECs. 39
I Effects of TMD23-wild type, TMD23 (I441A), TMD23 (D440A/D442A), and TMD23 (D440A/I441A/D442A) treatment on thrombin-enhanced permeability in HUVECs. 40

J Effects of TMD23-wild type, TMD23 (I441A), TMD23 (D440A/D442A), and TMD23 (D440A/I441A/D442A) treatment on thrombin-induced adhesion molecules expression in HUVECs. 41
K Effects of TMD23-wild type, TMD23 (I441A), TMD23 (D440A/D442A), and TMD23 (D440A/I441A/D442A) treatment on thrombin-induced MCP-1 in HUVECs. 42
L Effects of TMD23-wild type, TMD23 (I441A), TMD23 (D440A/D442A), and TMD23 (D440A/I441A/D442A) treatment on thrombin-induced TNFα in RAW264.7 cells. 44
M Effects of TMD23-wild type, TMD23 (I441A), TMD23 (D440A/D442A), and TMD23 (D440A/I441A/D442A) treatment on thrombin-induced proliferation in A7r5 cells. 45
N Animal model for the study of atherosclerosis 46
O Statistical analysis 48
XI. Results 49-52
XII. Discussions 53-56
XIII. References 57-60
XIV. Figures 61-76
1 Preparation and purification of mouse TMD23-wild type and three TMD23 mutant proteins. 61
2 Thrombin solid phase assay of TMD23-wild type and three TMD23 mutant proteins. 62
3 Thrombin dependent protein C activation assay of TMD23-wild type and three TMD23 mutant proteins. 63
4 Thrombin clotting time assay of TMD23-wild type and three TMD23 mutant proteins. 64
5 Effects of TMD23-wild type or TMD23 (I441A) treatment in thrombin-induced internalization of the surface PAR-1 on HUVECs. 65
6 Effects of TMD23-wild type, TMD23 (I441A), and TMD23 (D440A/D442A) treatment on thrombin-induced Ca2+ release in HUVECs. 66
7 Effects of TMD23-wild type and three TMD23 mutant proteins treatment on thrombin-enhanced permeability in HUVECs. 67
8 Thrombin induced adhesion molecules expression on HUVECs. 68
9 Time course of thrombin induced adhesion molecules expression on HUVECs. 69

10 Effects of TMD23-wild type and TMD23 mutant proteins treatment on thrombin-induced adhesion molecules expression in HUVECs. 70
11 Effects of TMD23-wild type and three TMD23 mutant proteins treatment on thrombin-induced MCP-1 in HUVECs. 71
12 Effects of TMD23-wild type and three TMD23 mutant proteins treatment on thrombin-induced TNFα in RAW264.7 cells. 72
13 Effects of TMD23-wild type and three TMD23 mutant proteins treatment on thrombin-induced proliferation in A7r5 cells. 73
14 Influence of TMD proteins treatment on neointima formation in C57BL/6 mice after carotid artery ligation. 74
15 Influence of TMD proteins treatment on atherosclerotic lesion formation in ApoE -/- mice. 75
16 Effects of TMD23-wild type (mammalian expression) treatment in thrombin-induced internalization of the surface PAR-1 and cytoplasmic Ca2+ release in HUVECs. 76
XV. Tables 77-78
1 Primer sequence 77
2 LC-MS protein identification 78
XVI. Appendixes 79-84
1 The structure of Thrombomodulin (TM). 79
2 Map of pPICZα-A and constructions of TMDs protein. 80
3 The four steps of Atherogenesis 81
4 Mouse carotid ligation surgery model 82
5 Interaction of the exosites (I and II) of thrombin in substrate recognition (depicted as yes or no) and with cofactors that accelerate the substrate reactions 83
6 Amino acid sequence of mouse thrombomodulin 84
XVII. Resume 85
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