臺灣博碩士論文加值系統

凝血酶調節素 (thrombomodulin，TM) 是一種廣泛表現的穿膜醣蛋白，具有多個功能區。除了抗凝血功能外，亦有研究指出凝血酶調節素參與調控發炎反應，但目前對於白血球上凝血酶調節素的功能尚未明瞭。本實驗室先前的研究顯示凝血酶調節素會透過其胺基端類凝集素功能區 (lectin-like domain) 特異性的結合醣類路易士Y (Lewis Y，Ley)，文獻指出路易士Y會透過醣化於黏附因子上，來調控細胞的黏附作用。因此，本論文主要探討在發炎時，白血球上凝血酶調節素是否會透過與內皮細胞上路易士Y的結合來調控白血球黏附。我們分析在多種發炎相關刺激物刺激情形下，人類單核球細胞 (THP-1) 上凝血酶調節素的表現量是否會受到影響，由實驗結果顯示經由巴豆醇-12-十四烷酸酯-13-乙酸酯 (phorbol 12-myristate 13-acetate)、腫瘤壞死因子α (tumor necrosis factor alpha) 及酯多醣體 (lipopolysaccharides) 刺激後，細胞表面的凝血酶調節素的表現量會上升。利用酵素連結免疫吸附法 (enzyme-linked immunosorbent assay)，發現游離態的路易士Y可以直接與細胞表面的凝血酶調節素結合。此外，滾動中的人類單核球細胞藉由凝血酶調節素黏附於流體腔上的路易士Y。我們更發現路易士Y和凝血酶調節素間的作用可以誘導p38的磷酸化，進一步促進β2整合素 (β2-integrin) 的活化。利用細胞穿孔實驗 (transwell assay) ，發現路易士Y和凝血酶調節素的結合可促進白血球細胞穿越活化的內皮細胞，並且此現象會被路易士Y和凝血酶調節素抗體所抑制。為了要觀察在動物模式中，白血球上凝血酶調節素是否會參與白血球趨化，我們利用頸動脈結紮手術誘發野生型(TMf/f)與白血球細胞上特異性凝血酶調節素剔除 (LysMCre/TMf/f) 小鼠的血管發炎反應，發現後者經由血管結紮後，白血球趨化和新生血管内膜增生較為減緩。綜合以上結果發現，在發炎情形下，白血球上凝血酶調節素會藉由與內皮細胞上路易士Y的結合來調控白血球和內皮細胞的交互作用。

Thrombomodulin (TM), a widely expressed transmembrane glycoprotein, has multiple functional domains. In addition to the well-known anticoagulant function, TM also regulates inflammatory responses. However, the role of TM on leukocyte still remains unclear. Our data previously showed that TM can specifically bind to the carbohydrate, Lewis Y (Ley), via its N-terminal lectin-like domain. Ley is found to be glycosylated on adhesion molecules and involved in cell adhesion. In this study, we intended to investigate whether leukocyte surface TM would participate in leukocyte adhesion by interacting with Ley on endothelial cells under inflammation. We analyzed membrane TM expression on THP-1 monocytic cells under various stimuli. The expression of TM on THP-1 monocytic cells was upregulated by monocyte activating agent, phorbol 12-myristate 13-acetate, as well as by inflammatory factors, tumor necrosis factor alpha and lipopolysaccharides. By using enzyme-linked immunosorbent assay, we found that soluble Ley could directly bind to TM on the surface of THP-1 monocytic cells. Moreover, the adhesion of rolling THP-1 monocytic cells to Ley-immobilized flow chamber was TM dependent. We further found that Ley could trigger p38 phosphorylation and was also TM dependent, which in turn led to β2-integrin activation, in THP-1 monocytic cells. By using transwell assay, the interaction of Ley and TM contributed to transendothelial migration, and which was abrogated by antibodies against either TM or Ley. To determine whether leukocyte TM would participate in leukocyte recruitment in vivo, we performed carotid artery ligation to induce vascular inflammation in wild-type (TMf/f) and myeloid-specific TM-deficient (LysMCre/TMf/f) mice. Reduced leukocyte recruitment and neointima formation were observed in myeloid-specific TM-deficient mice following carotid artery ligation. Our data provide a new insight showing that leukocyte TM could mediate leukocyte-endothelial interaction via binding to endothelial cell surface Ley under inflammation.

Chinese Abstract 1
Abstract 2
Acknowledgement 3
Content 4
Contents of Figures 5
Abbreviations 6
Instruments 8
Reagents and Chemicals 10
Introduction 14
Specific Aims 20
Materials and Methods 21
Results 41
Discussion 47
References 52
Figures 58
Resume 69

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