臺灣博碩士論文加值系統

凝血酶調節素 (Thrombomodulin, TM) 和內皮唾酸蛋白 (endosialin, ES) 屬於一新興的穿膜醣蛋白族群並且被分類在C型類凝集素功能區 (C-type lectin-like domain) 細胞表面蛋白的族群中。凝血酶調節素被認為是位在內皮細胞上作為蛋白質C抗凝血系統的輔因子。內皮唾酸蛋白原本被認為是癌症內皮細胞上的目標蛋白，後來又被認為是纖維母細胞上重要的辨識標記。內皮唾酸蛋白確切的功能到目前為止仍然不清楚。根據最近的研究指出，含有C型凝集素以及C型類凝集素功能區的細胞表面受器經常在免疫系統中扮演重要的角色。在本實驗室先前的研究中發現，利用重組的凝血酶調節素功能區片段2及3蛋白 (rTMD23) 可以在模擬動脈粥狀硬化的主動脈結紮動物模型中，有效的減少阻塞血管中的新生內層及中層面積比率，而動脈粥狀硬化目前被認為是一種與慢性發炎有關係的疾病。然而，關於重組的凝血酶調節素功能區片段2及3蛋白是否能在急性發炎中提供類似的保護效果則是不清楚的。在本實驗中，小鼠經由腹腔注射的方式接受脂多醣的刺激並接受重組的凝血酶調節素功能區片段2及3蛋白的治療。我們發現比起控制組，注射重組的凝血酶調節素功能區片段2及3蛋白能夠有效的改善存活率。為了釐清這樣的保護效果是透過活化態蛋白質C或者是凝血酶相關的路徑，我們製備了rTMD23-I424A (第424個胺基酸由I�莧) 以及rTMD23-D400A (第400個胺基酸由D�莧) 兩株突變蛋白，使它們分別在凝血酶結合能力以及蛋白質C活化作用受到突變的影響而造成活性下降，而這些突變的蛋白的抗發炎作用將會被進一步的研究。另外，在本實驗室先前的研究中發現重組的凝血酶調節素類凝集素功能區片段 (TMD1) 可以透過影響脂多糖與CD14的接合繼而達到抑制發炎的作用。在本實驗中，我們製備了內皮唾酸蛋白胺基端的C型類凝集素功能區蛋白 (endosialin domain 1, ESD1) 並對其進行類似的功能性探討，包括與脂多醣的結合能力、對於免疫細胞以及急性發炎動物模式的保護作用。我們發現內皮唾酸蛋白胺基端的C型類凝集素功能區能夠減少免疫細胞中腫瘤壞死因子α的釋出。除此之外，我們也發現注射內皮唾酸蛋白胺基端的C型類凝集素功能區蛋白能夠減少小鼠血清中的發炎性細胞激素的含量並且改善肺部發炎的情況。因此重組的凝血酶調節素功能區片段2及3蛋白以及內皮唾酸蛋白胺基端的C型類凝集素功能區蛋白都能改善脂多醣誘導的急性發炎的嚴重程度。我們認為內皮唾酸蛋白胺基端的C型類凝集素功能區蛋白是透過與脂多醣結合而抑制發炎反應路徑的活化，而重組的凝血酶調節素功能區片段2及3蛋白可能是透過與脂多醣接合無關的其他細胞保護路徑而達到減輕發炎的效果。

Thrombomodulin (TM) and endosialin (ES) belong to a newly described family of transmembrane glycoproteins and have been grouped into C-type lectin-like domain-containing cell surface proteins. TM is originally identified on endothelial cells and functions as a cofactor in the protein C anticoagulant system. ES was first described as a marker of tumor endothelium, and later as a marker of fibroblasts. The function of ES remains unknown. Recent studies suggest that C-type lectin and lectin-like receptors play an important role in the immune system. In our previous study, mice treated with recombinant TM domain 2 to 3 (rTMD23) have decreased intima/media ratio of the occluded vessels in aortic ligation model which mimics the pathogenesis of atherosclerosis, a chronic inflammatory disorder. It is unclear whether rTMD23 provides beneficial effects for acute inflammations. In this study, mice were treated with rTMD23 and lipopolysaccharide (LPS) via intravenous and intra-peritoneal administrations, respectively. We found that administration of rTMD23 improved survival of mice compared to control group. In order to clarify whether effects of rTMD23 were through activated protein C or thrombin, we constructed rTMD23-I424A and rTMD23-D400A which had reduced thrombin binding ability and activated protein C -generating ability respectively. The anti-inflammatory effects of rTMD23, rTMD23-I424A, and rTMD23-D400A were compared. In our previous study, N-terminal lectin-like domain of TM (TMD1) inhibits LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. In this study, lectin-like domain of endosialin (ESD1) was expressed and purified. LPS binding ability of ESD1 was examined. In vitro assays in THP-1 cells and the acute inflammation model mentioned above were also performed. We found that ESD1 decreased TNF-�� secretion in THP-1 cells. Besides, mice administrated with ESD1 had reduced serum cytokine and influx of inflammatory cells into lung. In conclusion, both rTMD23 and ESD1 reduced the severity of acute inflammation. The protection of ESD1 is through blockage of LPS-induced inflammatory signal pathway, while TMD23 may be through other cell protection pathways independent of LPS-blocking.

I.
Chinese Abstract 1
II.
Abstract 3
III.
Acknowledgments 4
IV.
Content Table 5
V.
Introduction
The C-type lectin - like domain (CTLD) containing proteins 8
Thrombomodulin (TM) 10
The role of TM in inflammation 10
The role of TM in atherosclerosis 13
Pathophysiology of sepsis 14
Endosialin 15
Specific aims 16
VI.
Materials and Methods
Part I. TM domain 2 to 3 (TMD23) 17
Quick-change site-directed mutagenesis 17
Expression of rTMD23, and mutant rTMD23 proteins from Pichia pastoris 20
Expression of rTMD23, and mutant rTMD23 proteins from mammalian cell HEK293 22
Purification of rTMD23, and mutant rTMD23 proteins 23
SDS-PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) 25
Silver staining 26
Western Blotting 27
Quantification of protein samples 28
Thrombomodulin activity assay 29
Thrombin binding activity assay 30
Effect of rTMD23 and mutant rTMD23 proteins on LPS-induced acute inflammation in vivo 31
Detection of Inflammatory mediators by ELISA (Enzyme-linked immunosorbent assay) 31
Myoperoxidase activity assay 33
Statistical analysis 33
Part II. N-terminal lectin-like domain of endosialin (ESD1) 34
Expression of ESD1 protein from Pichia pastoris 34
Purification of ESD1 protein 36
SDS-PAGE (Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis) 37
Silver staining 38
Western Blotting 39
Quantification of protein samples 40
LPS binding assay 41
THP-1 cell culture 41
Signal transduction analysis of THP-1 cells 42
Effect of ESD1 on LPS-induced Inflammatory mediators in THP-1 cells 43
Effect of ESD1 proteins on LPS-induced acute inflammation in vivo 43
Detection of Inflammatory mediators by ELISA (Enzyme-linked immunosorbent assay) 44
Myoperoxidase activity assay 45
ESD1 ligand analysis 46
Statistical Analysis 46
VII.
Result
Part I. TM domain 2 to 3 (TMD23) 47
Part II. N-terminal lectin-like domain of endosialin (ESD1) 49
VIII.
Discussion 52
IX.
Reference 56
X.
Figures and Legends
1 Purification of rTMD23 and mutant rTMD23 59
2 Cofactor activity of rTMD23 and mutated rTMD23 proteins 60
3 Thrombin binding activity of rTMD23 and mutated rTMD23 proteins 61
4 Effect of rTMD23 on lethality in LPS-induced inflammation in vivo 62
5 Effect of rTMD23 on inflammatory mediators production in vivo 62
6 Effects of rTMD23 on myeloperoxidase activities in lung 63
7 Purification of ESD1 64
8 LPS binding activity of ESD1 and rTMD23 protein 65
9 Effect of ESD1 on LPS-induced TNF-�� secretion in macrophage 65
10 Effect of ESD1 on LPS-induced signaling in macrophage 66
11 Effect of ESD1 on lethality in LPS-induced inflammation in vivo 67
12 Effect of ESD1 on inflammatory mediators production in vivo 67
13 Effects of ESD1 on myeloperoxidase activities in lung 68
14 Analysis of ESD1 ligand by AlphaScreen 69
XI.
Reagents, Drugs and Chemicals 70
XII.
Instruments 74
XIII.
Abbreviations 76
XIV.
Appendix
1 The structure of thrombomodulin 78
2 Vector map of pPICZ�哻 79
3 The DNA and protein sequence of thrombomodulin 80
4 The DNA and protein sequence of endosialin 84
5 Carbohydrate ligands panel 89
6 Primer of mutant rTMD23I424A 92
XV.
Resume 93

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