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系統識別號 U0026-0509201223470800
論文名稱(中文) 人類凝血酶調節素在小鼠角膜上皮細胞傷口癒合所扮演的角色
論文名稱(英文) The Role of Thrombomodulin at Corneal Epithelial Wound Healing in a Mouse Model
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 100
學期 2
出版年 101
研究生(中文) 陳俊嘉
研究生(英文) Chun-Chia Chen
電子信箱 chen.jica@msa.hinet.net
學號 s97961023
學位類別 碩士
語文別 英文
論文頁數 74頁
口試委員 召集委員-蔡曜聲
指導教授-吳華林
指導教授-謝式洲
中文關鍵字 人類凝血酶調節素  基因剔除鼠  傷口癒合  角膜 
英文關鍵字 Thrombomodulin  Transgenic mice  Wound healing  Cornea 
學科別分類
中文摘要 人類凝血酶調節素(Thrombomodulin,簡稱:TM)是原被發現位在血管內皮細胞表面的含有557個胺基酸的穿膜醣蛋白,目前研究已知其主要功能是調節血液的凝結反應和纖維蛋白的溶解有關。結構上被區分為五個功能區:第一個功能區是氮端的154個氨基酸構成"類似lectin的功能區"(lectin-like domain),因於該區域的胺基酸排列類似於C-型lectin(C-type lectin);第二個功能區是由六個表皮生長因子類似區(epidermal growth factor modules;EGF modules)所組成的"類似EGF功能區"(EGF-like domain);第三個功能區是富含絲胺酸/酥胺酸區(serine/theronine-rich region),主要為蛋白質醣基化(包含N-,O-glycosylation)的部位;接著第四、第五功能區分別是穿膜功能區(transmembrane domain)以及細胞質功能區(cytoplasmic dom-ain)。當TM 與凝血酶(thrombin)結合形成複合體時,可以活化Protein C,而被活化的Protein C 可以分解凝血因子Ⅴa 與Ⅷa,中斷凝血機制。然而凝血酶調節素除了表現在內皮細胞上之外,也會表現在其它的細胞,像是皮膚的角質細胞等。有研究發現小鼠的凝血酶調節素基因剔除後,小鼠的胚胎僅能發育至第8.5天就會終止生長,其終止生長的原因並不是因為血栓功能失調,故推論於正常的胚胎發育中,凝血酶調節素可能還扮演了抗凝血以外的角色。而目前也有研究指出,凝血酶調節素在上皮細胞增生與分化時,會大量的表現,特別是在傷口癒合的時期。雖然傷口癒合的機轉相當的複雜,包含了凝血,發炎反應,肉芽組織的增生,上皮細胞的生長,以及傷口的穩定。而且過程中的每一個步驟,都會深深影響一個正常的傷口癒合。

上皮細胞的生長促進傷口癒合的過程,通常在傷口一旦形成的時候就開始了,而且為傷口癒合的重要步驟之一。在臨床上,糖尿病人常見傷口癒合有明顯的困難。同時,我們也發現,這類的病人會有人類凝血酶調節素異常的現象。此外,在一些特殊的傷口癒合狀況,例如蟹足腫疤痕,也會出現人類凝血酶調節素異常的現象。根據以前的研究,發現凝血酶調節素第二及第三結構體(Domain), TMD23,與細胞的增殖及血管增生有關。 根據許多的文獻,發現角膜上皮細胞的癒合生長,和許多的生長激素有關,其中凝血酶(Thrombin)參與的機轉有關,但是關於人類凝血酶調節素是否參與,鮮少有研究說明。唯有少量的研究指出在角膜癒合的過程,發現有人類凝血酶調節素的出現。本實驗室構築出可以製造人類TMD23 蛋白的酵母菌,利用TMD23 蛋白與特異組織人類凝血酶調節素剔除鼠(基因剔除鼠),再進一步的對凝血酶調節素對角膜上皮細胞傷口癒合的特性和功能做進一步的分析與研究。
英文摘要 Thrombomodulin(TM) is an important glycosylated transmembrane molecule of 557 amino acids that regulates coagulation and fibrinolysis. TM consists of 1. an N-terminal globular domain with weak sequence similar to C-type animal lectins, 2.an extended stalk of six EGF modules, 3.a serine/threonine–rich region with target sites for post-translational N- and O-glycosylation, 4.a transmembrane domain, and 5. a short cytoplasmic tail. It is well known that EGF domains 5 and 6 bind thrombin. EGF domain 4 is required for Protein C activation. However, TM is not only expressed in endothelial cells but also in epidermal keratinocytes. Knockout of the TM gene in mice results in embryonic lethality independent of thrombosis, suggesting that structures of TM might play important roles in normal fetal development rather than anticoagulation function.

Expression of TM by epidermal keratinocytes is tightly regulated during squamous cells differentiation and cutaneous wound healing. Although cutaneous wound healing is a complicated process involves a series of sequential stages including coagulation, inflammation, formation of granulation tissue and wound remodeling. Re-epithelialization begins as soon as the wound damages firstly and is required for the reestablishment of the skin. In clinical observations, TM deficiency in local wound was showed in diabetic patients. They appear to have a strongly relationship in the wound healing process. The abnormal expression of TM was found in keloid scar and hypertrophic skin. Although the fragment of thromobomodulin is proven to have a strong angiogenic effect, the relationship between re-epithelialization and TM is still not clear. Thus, the role of TM in corneal epithelial wound healing process was interesting to be investigated. By comparison of corneal epithelial wound healing, keratinocyte migration and re-epithelialization were observed between TM tissue-specific deletion mice (transgenic, TMLox/Lox K5-Cre mice) and wild type mice. Transgenic mice exhibit a significant delay in wound re-epithelialization.
論文目次 I Chinese abstract 中文摘要 1-2
II Abstract 英文摘要 3-4
III Acknowledgments 誌謝 5-7
IV Content table 目錄 8
V Tables of Figures and Legends 圖目錄; 附錄目錄 9; 10
VI Abbreviation table 符號 11-12
VII Introduction 引文 13-20
VIII Materials and Methods 材料與方法 21-24
IX Results 結果 25-26
X Discussion 討論 27-33
XI References 參考文獻 34-38
XII Figure and Legends圖表 39-48
XIII Instruments 儀器 49
X IV Reagents 藥劑 50
XV Appendixes 附錄 51-71
X VI Person's resume 個人簡介 72
X VII Publications 論文著作 73-74
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