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  • 學位論文

基質金屬蛋白酶-7水解凝血調節素之衍生片段參與表皮細胞株之表皮-間葉過渡研究

MMP-7 mediated proteolytic processing Thrombomodulin involved in Epithelial-Mesenchymal transition (EMT)

指導教授 : 游偉絢
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摘要


凝血脢調節素(TM)最初發現於血管壁內皮細胞表面的一種醣蛋白,初期此蛋白質具有天然內皮層抗凝血素之特性。不過,近年來的研究著重於凝血脢調節素的表現量與上皮細胞的上皮-間質轉化(EMT)兩者的關聯性。為了研究基質金屬蛋白酵素7(MMP-7)在上皮細胞進行上皮-間質轉化時,凝血脢調節素受蛋白質水解時可能扮演的角色,本實驗建立了一個共同轉染有凝血脢調節素與基質金屬蛋白酵素7兩者表現質體的細胞株(MDCK)。利用西方墨點分析法確認了凝血脢調節素與基質金屬蛋白酵素7的表現量後,觀察到一個有趣的結果,即在共轉染有凝血脢調節素與基質金屬蛋白酵素7兩基因的MDCK細胞中,凝血脢調節素會受到金屬水解酵素所切割。另外,從核質分離試驗中,本實驗發現了凝血脢調節素片段中的一個分子量約為34 kDa的蛋白質片段,的確存在於細胞核內,從共軛焦顯微鏡之影像分析結果,也確認了此凝血脢調節素片段確實會分布於細胞核。   在胚胎發育過程中會發生上皮-間質轉化現象,而與其相關之腫瘤形成之初,也具有此轉換之現象。在上皮-間質轉化的過程之中,SNAIL蛋白的表現量與腫瘤細胞的移動性與侵犯性之強弱有很高的關聯性。從初步的實驗結果看來,在MDCK細胞株中,基質金屬蛋白脢7所切割後而形成之人類凝血調節素片段,會與SNAIL蛋白進入細胞核機制有關,進而調控下游各種基因的表達,包括凝血調節素本身基因。然而,SNAIL蛋白與細胞質中的凝血脢調節素片段如何形成複合體,進而轉移到細胞核中和其背後所代表的生物功能仍需被釐清。

並列摘要


Thrombomodulin (TM) is a glycoprotein that was originally identified in vascular endothelium and characterized as a natural endothelial anticoagulant [1]. From recent studies[2] [3] [4], it is focusing on the role of TM during Epithelial-mesenchymal transition (EMT). In this study, we investigated the potential role of MMP-7 in proteolysis of TM protein during EMT. An epithelial cell line, MDCK, was transfected with TM and/or MMP-7. Using western blotting analysis the expression of MMP7 proteins could make TM being proteolytically processed with formation of multiple fragments of TM in the TM and MMP7-cotransfected MDCK. Furthermore, from the nuclear v.s. cytoplasmic fraction assay, we demonstrated that the major ~34 kDa fragment was seen in the nuclear fraction. Confocal microscopy image analysis also further confirmed the nuclear localization of TM fragments. Epithelial-mesenchymal transition (EMT) occurs during embryonic development and may also be responsible for onset of EMT-associated tumorigenesis. During EMT, SNAIL protein is up-regulated and its expression levels are strongly correlated with increased motility and invasion of cells [5] [6]. From the preliminary results, we found that the processing of human thrombomodulin protein by MMP-7 in MDCK cells, which may be associated with translocation of SNAIL protein into nucleus to regulate its target genes [7], including TM gene activation. However, the biological functions of nuclear-translocation of the ternary complex of SNAIL and cytosol TM are still unclear and need to be further elucidated in the future.

並列關鍵字

MMP-7 Thrombomodulin EMT

參考文獻


[1] Pekovich, S.R., Bock, P.E. and Hoover, R.L. (2001). Thrombin-thrombomodulin activation of protein C facilitates the activation of progelatinase A. FEBS Lett 494, 129-32.
[3] Przybylo, J.A. and Radisky, D.C. (2007). Matrix metalloproteinase-induced epithelial-mesenchymal transition: tumor progression at Snail's pace. Int J Biochem Cell Biol 39, 1082-8.
[4] Jorda, M., Olmeda, D., Vinyals, A., Valero, E., Cubillo, E., Llorens, A., Cano, A. and Fabra, A. (2005). Upregulation of MMP-9 in MDCK epithelial cell line in response to expression of the Snail transcription factor. J Cell Sci 118, 3371-85.
[5] Barbera, M.J. et al. (2004). Regulation of Snail transcription during epithelial to mesenchymal transition of tumor cells. Oncogene 23, 7345-54.
[6] Yokoyama, K., Kamata, N., Fujimoto, R., Tsutsumi, S., Tomonari, M., Taki, M., Hosokawa, H. and Nagayama, M. (2003). Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas. Int J Oncol 22, 891-8.

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