臺灣博碩士論文加值系統

凝血酶調節素(thrombomodulin, TM)是一個表現於血管內皮細胞表面的穿膜蛋白，在生理上是具有抗凝血功能的重要因子。 而近年來TM被發現也表現於許多腫瘤細胞上，同時也發現TM表現量的多寡和腫瘤的惡性程度呈現負相關；這暗示著TM可能具有抑制腫瘤惡化的作用。 此外，TM也被發現能以游離狀態(soluble form)存在於血液和尿液中，這種游離形式的TM能夠有效地抑制腫瘤細胞的侵襲爬行和轉移，而這樣的抑制效果與其抗凝血功能無關，但卻需要TM的類凝集素功能區(lectin-like domain)參與其中的調控。 本篇研究發現，TM的類凝集素功能區(TMD1)具有能夠抑制腫瘤轉移的作用。 實驗結果顯示，TMD1並不具有細胞毒性，不會引起細胞凋亡，但卻能夠有效地抑制小鼠黑色素瘤細胞B16F10的侵襲爬行，且其抑制效果和施加劑量呈現正相關。 同時，TMD1能夠降低MMP-2活性及其mRNA表現量，並且也能抑制MMP-2上游訊號的活化，如：ERK1/2及Akt。 此外，使用螢光標定的方式顯示，TMD1能夠專一性地黏附於細胞膜上，或許因此影響了細胞的行為表現。 而一個可能的目標是CXCR4 (C-X-C chemokine receptor 4)，因為加入CXCR4抗體和CXCR4的配體CXCL12均能夠有效地競爭TMD1與細胞的黏附。 TMD1也確實能夠抑制由CXCL12所引起的腫瘤細胞爬行和MMP-2活性。 另外，TMD1所具有的抑制效果不止是針對黑色素瘤細胞，對於其他腫瘤，如：乳癌和肺癌，同樣也具有明顯的抑制作用。 這些實驗結果顯示，TMD1擁有能作為一個抑制癌細胞轉移之藥物潛能，並且對於多種癌細胞皆能發生作用。

Thrombomodulin (TM) is an integral membrane protein expressed on the endothelial cell surface and first known as an anticoagulant factor. Recently, it is reported that TM is also expressed on the plasma membrane of several tumor cells and the expression level of TM is negatively correlated with cancer malignancy. It was further implicated that TM functions as a negative regulator in tumor progression. Furthermore, soluble TM existing in plasma could suppress tumor invasion and metastasis independent of its anticoagulant activity but involved in its lectin-like domain. In this study, we demonstrated that the lectin-like domain of TM (TMD1) might contribute to the antitumoral function. The invasiveness of murine melanoma B16F10 cells was significantly inhibited by TMD1 in a dose-dependent manner. This inhibitory effect was not due to cytotoxicity of TMD1 because TMD1 didn’t change the cell growth rate. TMD1 could dose-dependently decrease both the proteolytic activity and the mRNA expression of matrix metalloproteinase-2 (MMP-2) in B16F10 cells. Additional, TMD1 reduced the activation of signal pathways related to MMP-2 expression, such as ERK1/2 and Akt signaling. Moreover, rhodamine-labeled TMD1 could bind to B16F10 cells dose- dependently and the binding could be competed by either TMD1 specific antibody or cold TMD1, suggesting that TMD1 might bind to some specific receptors and interfere with the downstream signaling transduction. We identified that one of the possible candidates is C-X-C chemokine receptor 4 (CXCR4)/C-X-C chemokine ligand 12 (CXCL12) axis because TMD1 could inhibit CXCL12-induced MMP-2 activity and cell invasion. Furthermore, the inhibitory effects of TMD1 on MMP-2 activity and ERK1/2 activation could also be observed in different tumor cell types such as A549 (human lung cancer cells) and MDA-MB-231 (human breast cancer cells). The results suggested that TMD1 could be a novel therapeutic antimetastatic drug for various tumor types.

Introduction ………………………………………………………1
Materials and Methods ……………………………………………4
Expression and Purification of Recombinant TM domains ……………… 4
Gel Electrophoresis and Western Blotting ………………………………… 6
Gel Staining ……………………………………………………………… 9
Cell Lines and Culture Conditions ……………………………………… 11
Cell Invasion Assay ………………………………………………………… 12
Cell Transendothelial Migration Assay ………………………………… 14
Cell Proliferation Assay …………………………………………………… 15
Zymography Assay ………………………………………………………… 16
RNA Extraction, RT-PCR and Real-time PCR analysis ………………… 18
Flow Cytometry Assay …………………………………………………… 21
Statistic Analysis …………………………………………………………… 23
Results ………………………………………………………………24
Discussion ……………………………………………………………28
Figures ………………………………………………………………32
References ……………………………………………………………45
Appendixes ……………………………………………………………50
Author’s File ………………………………………………………60

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