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系統識別號 U0026-0812200915221087
論文名稱(中文) 製備人類凝血酶調節素類表皮生長因子結構區之重組蛋白及其功能分析
論文名稱(英文) Preparation and Functional Analyses of Various Recombinant Thrombomodulin Derived Epidermal Growth Factor (EGF)-like Structures
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 97
學期 2
出版年 98
研究生(中文) 鄭又銘
研究生(英文) Yu-Ming Cheng
電子信箱 s1696501@mail.ncku.edu.tw
學號 s1696501
學位類別 碩士
語文別 英文
論文頁數 73頁
口試委員 口試委員-林淑華
口試委員-張文粲
口試委員-施桂月
指導教授-吳華林
中文關鍵字 EGF-like 結構區  凝血酶調節素 
英文關鍵字 Thrombomodulin  epidermal growth factor (EGF)-like structure 
學科別分類
中文摘要 凝血酶調節素 (thrombomodulin, TM) 是一種表現於血管內皮細胞表面的醣蛋白,是生理上重要的抗凝血因子,其蛋白質結構從氨基端依序被分為類外源凝集素(lectin-like)結構區(TMD1), EGF-like 結構區(TMD2), serine/thrtonine-rich 結構區(TMD3), transmembrane 結構區(TMD4) 及碳端的cytoplamic結構區(D5)。凝血酶調節素在不同組織中扮演著:抑制轉移、血管新生調節素、細胞間的連結並具有在不同組織間的抗發炎反應。在之前的研究中,我們發現TMD23重組蛋白,不論是在體內或是體外實驗,都能促進細胞的增生,移動,以及血管的新生。TMD23 重組蛋白,有效改善了K5-Cre/TMLox/Lox老鼠的傷口癒合情形。至於是哪一個EGF結構區或是因為不同凝血酶調節素,在醣化程度功能上的影響與否,仍然有待研究。在這個研究中,我們利用哺乳類表達系統呈現不同的D2以及D3結構區重組蛋白,分別為TMD2 (EGF1-6) D3, TMD2 (EGF2-6) D3, TMD2 (EGF3-6) D3, TMD2 (EGF4-6) D3, TMD2 (EGF5-6) D3 以及TMD2 (EGF6) D3。每一個重組蛋白都可以純化出兩種不同的醣化型:高分子量以及低分子量重組蛋白。高分子量重組蛋白經過chondroitinase ABC酵素作用後,其分子量即轉變成低分子量重組蛋白。接著我們更進一步證實,高分子量TMD23重組蛋白具有與低分子量TMD23重組蛋白相似的生物活性。高分子量重組蛋白不論在與凝血酶(thrombin)結合、活化蛋白質C或是延長凝血酶引起的凝血時間,對人類臍靜脈內皮細胞(human umbilical vein endothelial cell) 的生長,移動和類似毛細血管生成的調節也比低分子量重組蛋白來的有效。除此之外,高分子量重組蛋白在刮傷癒合(scratch wound healing assay)以及老鼠傷口癒合的分析中,其促進傷口癒合的速度也比低分子量重組蛋白來的迅速。在敗血症研究的動物模式中,我們也發現高分子量重組蛋白可以抑制LPS所引起的TNF-α 及IL-6的釋放。綜合以上的實驗結果,顯示高度醣類修飾的凝血酶調節素重組蛋白,在抗凝血與傷口癒合上具有較強的生物活性,也降低敗血症引起的死亡率,這意味著在臨床藥物上的開發,是具有潛在的實用價值。
英文摘要 Thrombomodulin (TM) is an endothelial cell surface anticoagulant glycoprotein is composed of five distinct domains contain 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) that perform antimetastatic, angiogenic, adhesive, and anti-inflammatory functions in various tissues. In our previous study, the recombinant soluble human TM domains 2 and 3 (TMD23) promoted cell proliferation, migration and angiogenesis. The TMD23 significantly improved wound closure in the K5-Cre/TM Lox/Lox mice. However, which epidermal growth factor (EGF)-like structure and whether the presences of carbohydrate were critical for TM functions remains unclear. Human embryonic kidney 293 mammalian expression system was used to prepare various TM domain 2 (D2) plus domain 3 (D3) including TMD2 (EGF1-6) D3, TMD2 (EGF2-6) D3, TMD2 (EGF3-6) D3, TMD2 (EGF4-6) D3, TMD2 (EGF5-6) D3 and TMD2 (EGF6) D3. Each TM mutant contains two glycoforms [high molecular weight (high-Mw) and low molecular weight (low- Mw)]. The high-Mw TMDs were converted into low-Mw TMDs by chondroitinase ABC digestion. The high-Mw TMDs had higher ability to bind thrombin, to activate protein C, prolonged clotting time in a dose-dependent fashion, and had higher activity to promote human umbilical vein endothelial cell (HUVEC) proliferation and migration than low-Mw TMDs. High-Mw TMDs enhanced HaCaT wound healing in vitro and re-epithelialization during wound healing in vivo. The high-Mw proteins were administrated intravenously in mice 30 min before infusing LPS (lipopolysaccharide). The result showed that high-Mw protein dramatically inhibited LPS-induced serum TNF-α脉 and IL-6 elevation. In conclusion, these studies suggest that the extent of glycosylation had profound effects on the anticoagulant and skin wound healing properties of human TM. High-Mw TM also reduced mortality in sepsis animal model. We suggest that high-Mw TM proteins have potential to become useful therapeutic agents in the future.
論文目次 I. Abstract in Chinese 1
II. Abstract in English 2
III. Acknowledgement 3
IV. Contents 4
V. Contents of figures and tables 6
VI. Abbreviations 7
VII. Reagents 9
VIII. Instruments 13
IX. Introduction 15
A Anti-coagulation 17
B Angiogenesis 17
C Wound healing 18
D Pathophysiology of Sepsis 18
X. Specific Aim 19
XI. Materials and Methods
A Polymerase chain reaction (PCR) 20
B Restriction enzyme digestion 20
C Gel extraction 21
D Ligation 22
E Transformation of E. coli 22
F Plasmid DNA Extraction 23
G Agarose Gel Electrophoresis 24
H HEK293 transfection and selection 24
I Expression of recombinant thrombomodulin
with HEK293 26
J Anion exchanger chromatography 26
K Affinity Ni2+-chelating Sepharose
chromatography 27
L Cell Culture 28
M Western Blotting 30
N Chemotaxis assay (Boyden chamber) 33
O Cell Proliferation Assay 34
P In vitro Matrigel Angiogenesis Assay 35
Q Thrombomodulin Activity Assay 35
R Thrombin Solid-phase Assay 37
S Thrombin Clotting Time 38
T Scratch Wound Healing Assay 38
U Mouse skin wound healing 39
V Animal model of LPS-induced acute
inflammation 39
W Detection of inflammatory by ELISA 40
XII. Results 42
XIII. Discussion 47
XIV. References 51
XV. Figures
1 Preparation and purification of various
human TM domains with HEK293 based
mammalian expression system. 56
2 Western blot analysis and schematic
representation of recombinant human TM 57
3 Effects of chondroitinase ABC on low-Mw and
high-Mw TMD23 cofactor activity. 58
4 Thrombin solid phase assay of low-Mw and
high-Mw TMD23 and various EGF-like
structures. 59
5 Thrombomodulin protein C cofactor
activity. 60
6 Effect of low-Mw and high-Mw TMDs on
thrombin clotting time (TCT) assay. 61
7 Effects of low-Mw and high-Mw TMD23 on
HUVECs proliferation and migration. 62
8 Effect of tube formation of HUVECs on
Matrigel in vitro. 63
9 TMD23 enhance wound healing in vitro 64
10 Enhancement of skin wound healing in
K5-Cre/TM Lox/Lox mice with low-Mw and
high-Mw TMD23 treatment. 65
11 Effect of TMD23 on inflammatory mediator
production in vivo. 66
12 Effect of TMD23 on lethality of LPS-induced
inflammation in vivo. 67
XVI. Tables
1 Primer sequence of various TMD23 68
2 Bioinformation of various recombinant TMD23
proteins 69
XVII. Appendixes
1 Structure of thrombomodulin 70
2 Bioinformation of recombinant TMD
proteins 71
3 Amino acid sequence of each EGF like human
thrombomodulin 72
XVIII. Resume 73
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