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系統識別號 U0026-0812200911123757
論文名稱(中文) 探討凝血酶調節素在傷口復原時扮演的角色
論文名稱(英文) The role of thrombomodulin in wound healing process
校院名稱 成功大學
系所名稱(中) 生物化學研究所
系所名稱(英) Department of Biochemistry
學年度 92
學期 2
出版年 93
研究生(中文) 陳永煥
研究生(英文) Yung-Huan Chen
電子信箱 afran9@ms40.url.com.tw
學號 s1691108
學位類別 碩士
語文別 中文
論文頁數 91頁
口試委員 指導教授-施桂月
口試委員-趙曉秋
口試委員-吳華林
口試委員-林淑華
中文關鍵字 角質細胞  皮膚  傷口復原  凝血酶調節素 
英文關鍵字 cutaneous  keratinocyte  wound healing  thrombomodulin 
學科別分類
中文摘要   凝血酶調節素(thrombomodulin;簡稱TM)是一種位於細胞表面,由557個胺基酸所組成的醣蛋白,其主要功能是調控血液凝集以及纖維蛋白溶解反應。結構上被區分為五個功能區:氮端的154個氨基酸構成"似lectin功能區"(lectin-like domain),起因於該區域的胺基酸排序類似於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 fact- or modules;EGF modules)所組成的"似EGF功能區"(EGF-like domain);富含絲胺酸/酥胺酸區(serine/theronine-rich region)屬於第三個功能區,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)。

  凝血酶調節素的表皮生長因子類似區(epidermal growth factor-like mo- dules;EGF modules)是主要提供抗凝功能的區域,其中EGF4-6調節區包含重要的鈣離子結合位,也是非共價結合凝血酶、活化protein C的部位;EGF3則是凝血酶-凝血酶調節素複合物結合thrombin-activatable fibrinolysis inhibitor(TAFI)的區域。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上。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天便終止生長,其死因並非由於血栓失調所造成,故推論凝血酶調節素於正常的胚胎發育中可能還扮演了抗凝血以外的角色。

  為了探討凝血酶調節素在表皮層(epithelium)的功能,本實驗室建立了兩種動物模式:大鼠視網膜受傷模式(rat retinal injury model)以及小鼠皮膚受傷模式(mouse cutaneous excision wound model)。在大鼠視網膜受傷模式中發現視網膜受傷後,凝血酶調節素基因的表現並沒有明顯的改變;而在小鼠皮膚受傷模式中發現,在傷口周圍的凝血酶調節素基因表現量在受傷後第一天就會提高,在第三至第四天達到最高的表現量接著凝血酶調節素基因的表現會慢慢降低並在約第九天恢復到正常表現量。再藉由組織免疫染色近一歩發現凝血酶調節素會大量的表現在新生的表皮上。於另一方面以基因工程製備的重組蛋白rTMD23(包含表皮生長因子調節區和富含絲胺酸/酥胺酸功能區)作用於角質細胞上,進行相關研究發現rTMD23能增加角質細胞的移動(migration)。由於角質細胞的移動是皮膚傷口癒合過程中的一個重要步驟,故進一步我們將凝血酶調節素的片段rTMD23當作敷料,應用在小鼠受傷的皮膚上,觀察皮膚傷口癒合的情形是否有所改變;實驗結果顯示rTMD23對於皮膚傷口的閉合(wound closure)有些微促進;對於表皮再生(reepithelialization)則無顯著差異。但是在受傷後第十二天觀察到rTMD23有助於減少傷口水蒸氣的蒸散,其代表rTMD23能促進傷口癒合之速度。此外,亦將蟹足腫(keloid)、肥厚性疤痕(hypertrophic scar)、家族良性慢性天疱瘡(Hailey-Hailey disease)、毛囊角化病(Darier´s disease)四種皮膚病病人的皮膚切片藉由組織免疫染色觀察發現凝血酶調節素的表現量以及分布位置皆和正常皮膚不同。



英文摘要   Thrombomodulin (TM) is a glycosylated transmembrane molecule of 557 amino acids that regulates coagulation and fibrinolysis. TM consists of an N-terminal globular domain with weak sequence similarity to C-type animal lectins, an extended stalk of six EGF modules, a serine/threonine–rich region with target sites for post-translational N- and O-glycosylation, a transmembrane domain, and 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. For thrombin-activatable fibrinolysis inhibitor interaction, EGF module 3 is also required. TM, however, 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 other important roles in normal fetal development.

  In order to study the role of TM in the epithelium, we set up two animal models, the rat retinal injury model and mouse cutaneous excision wound model. In the rat retinal injury model, TM expression level was not induced by injury. In the mouse cutaneous excision wound model, TM expression levels in the skin was upreglated on day 2 after wound was created, and retuned to normal on day 9. On day 3 after wound was created, strong immunohistochemical staining of TM was observed in keratinocytes near the region of neoepidermis. In vitro test, we found that recombinant TM domain 2 and 3( rTMD23 ), including six EGF modules and serine/threonine–rich region, could induce keratinocyte migration. Since keratinocyte migration is an important step in cutaneous wound healing, we further tested the effect of rTMD23 on wound healing. The wounds treated with rTMD23 showed slightly faster wound closure and reepithelialization , but had no statistical significance. The skin evaporated rate after wound was reduced in rTMD23- treated group, suggesting rTMD23 might improve wound healing. In immunohistochemical localization analysis the normal human skin showed that TM expressed restrictly in the stratum spinousum of epidermis.It’s interesting that the patients with keloid, hypertrophic scar, Hailey-Hailey disease and Darier´s disease, we found that TM expressed not only in the stratum spinousum but also stratum basale and stratum granulosum.



論文目次 目錄 頁次
中文摘要------------------------------------------------------------------------1
英文摘要------------------------------------------------------------------------3
誌謝----------------------------------------------------------------------------5
目錄----------------------------------------------------------------------------6
圖、表、附錄目錄----------------------------------------------------------------8
縮寫檢索表---------------------------------------------------------------------10
儀器---------------------------------------------------------------------------11
藥品---------------------------------------------------------------------------13
緒論---------------------------------------------------------------------------16
一、人類凝血酶調節素概論---------------------------------------------------16
二、人類凝血酶調節素的結構-------------------------------------------------17
三、人類凝血酶調節素的分布和功能-------------------------------------------18
四、皮膚構造概論-----------------------------------------------------------19
五、皮膚傷口癒合過程-------------------------------------------------------20
六、研究目的與動機---------------------------------------------------------23
實驗方法與材料-----------------------------------------------------------------24
一、凝血酶調節素-聚合酶連鎖反應(TM-PCR)
動物組織RNA的萃取------------------------------------------------------24
DNase treat------------------------------------------------------------25
反轉錄-聚合酶連鎖反應(RT-PCR)------------------------------------------26
TM and β-actin -聚合酶連鎖反應(TM and β-actin-PCR)-------------------27
RNA濃度測定------------------------------------------------------------29
瓊脂膠電泳分析(agarose gel electrophoresis)----------------------------29
二、細胞培養方法
人類皮膚角質細胞(HaCaT)之冷凍細胞活化------------------------------------------30
人類皮膚角質細胞(HaCaT)之細胞計數--------------------------------------31
人類皮膚角質細胞(HaCaT)之繼代培養--------------------------------------32
凍細胞的方法-----------------------------------------------------------32
三、Migration assay(Boyden chamber)----------------------------------------33
四、大鼠動物實驗(rat retinal injury model)
大鼠視網膜傷害手術-----------------------------------------------------35
五、小鼠動物實驗(mouse cutaneous excision wound model)
小鼠脫毛、分組---------------------------------------------------------35
皮膚傷口手術&rTMD23處理傷口--------------------------------------------36
測量上皮再生百分比&傷口閉合百分比--------------------------------------37
測量角質細胞移動距離---------------------------------------------------38
測量皮膚水蒸氣蒸散情形-------------------------------------------------38
六、組織染色及切片
玻片前處理-------------------------------------------------------------39
石臘包埋---------------------------------------------------------------39
蘇木紫&伊紅染色(HE stain)----------------------------------------------40
Massion’s trichrome stain---------------------------------------------41
免疫組織染色(Immunohistochemical staining)-----------------------------43
結果---------------------------------------------------------------------------46
討論---------------------------------------------------------------------------50
參考文獻-----------------------------------------------------------------------53
結果圖表-----------------------------------------------------------------------59
表-----------------------------------------------------------------------------79
附錄---------------------------------------------------------------------------80
自述---------------------------------------------------------------------------91
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