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研究生：	邵鈞義 Shau, Jiun-Yi
論文名稱：	Gelsolin在口腔癌細胞侵犯,貼附和局部移動之研究 A study in the function of gelsolin in oral cancer invasion adhesion and local motion
指導教授：	謝達斌 Shieh, Dar-Bin 黃溫雅 Huang, Wen-Ya
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	58
中文關鍵詞：	間質金屬蛋白酶、肌動蛋白、口腔癌、膠原蛋白、胞外基質、細胞移動、轉移、細胞貼附、腫瘤、組織侵犯、細胞骨架
外文關鍵詞：	actin filament, matrix metalloproteinases, AFM, collagen, cytoskeleton, cell motility, oral cancer, tumor, metastasis, gelsolin, cell invasion
相關次數：	點閱：201 下載：3
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　　我們所研究的gelsolin 是一種肌動蛋白(actin) 細胞骨架(actin filament)的調控蛋白，而細胞型態的維持,細胞的移動和侵犯等細胞行為,都需要藉由細胞骨架的重組還達成。gelsolin可在肌動蛋白細胞骨架上進行切割,套蓋和成核等作用來造成細胞骨架的重新排列，而且gelsolin的活性會受鈣離子濃度，PIP2分子以及酸鹼值所調控。在本實驗室過去的研究中發現利用免疫化學染色技術比較口腔癌癌前病變和口腔癌晚期的組織切片gelsolin的表現情形，我們發現在口腔癌癌前病變的組織中gelsolin是屬於低度表現的，相反的,口腔癌晚期gelsolin的表現情形則有上升的現象。局部侵犯和遠端轉移是造成口腔癌治療失敗的重要原因之一，因此我們這次的研究重點在於探討口腔癌細胞進行細胞侵犯，細胞移動以及貼附等功能時，gelsolin所扮演的角色。過去由許多論文發表中得知gelsolin的表現具有促進細胞侵犯的現象，而在我們的研究中利用細胞侵犯實驗(matrigel invasion assay)和細胞侵犯膠原蛋白實驗(collagen gel invasion assay)也證實了gelsolin高表現量的細胞株具有較強的侵犯能力。造成細胞侵犯的要素大致可就由幾個方向來討論：(1)細胞分泌物質(如間質金屬蛋白酶)分解胞外基質(extracellular matrix)的能力，(2)是否改變細胞移動(cell motility)的速度，(3)細胞貼附(cell adhesion)胞外基質的能力， (4)細胞吞噬胞外基質的能力(phagocytosis)。我們發現gelsolin表現量的高低對於細胞移動的速度和基質金屬蛋白酵素的活性上並沒有明顯的差異。但在塗佈膠原蛋白的培養皿上進行細胞貼附測試，發現gelsolin 高表現量的細胞株相較於低表現gelsolin的細胞株有較強的貼附能力。此外利用細胞聚集實驗(slow and fast cell aggregation assay)發現gelsolin 高表現量的細胞株只能形成較小且較零散的細胞團塊，而相對於較低表現量的細胞株則可形成較大且集中的細胞聚集。而之後利用雞心組織細胞侵犯實驗(chick heart invasion assay)，更接近於in vivo的情況下比較gelsolin對於口腔癌細胞侵犯能力的探討，從結果可證實gelsolin的高表現量會促進癌細胞侵犯組織。此外，我們利用原子力顯微鏡比較在不同gelsolin表現量的細胞中，其細胞表面結構上的差異、力學及彈性上的變化，得知在gelsolin高表現量的細胞除具有較高的彈性和柔軟度，亦發現細胞在塗佈膠原蛋白的基材具有較高的貼附能力，這與細胞貼附實驗中得到的結果相符。綜合以上的實驗結果，我們認為在口腔癌細胞株中gelsolin具有促進細胞侵犯的現象，但促進侵犯的結果並非藉由增加細胞移動能力和胞外基質分解酶的活性，可能是因為gelsolin表現增加使細胞和細胞之間的貼附作用減弱，並加強了細胞對於胞外基質的附著能力。也就是指癌細胞容易脫離原來所在的細胞團，深入進而穿越胞外基質到達血液中，再運送到其他組織，造成細胞侵犯現象。

　Regional invasion and distant metastasis are important factors for clinical failure in oral cancer therapy. Dynamic changes in cell morphology, motility and invasion into extracellular matrix were known to involve the cell invasive process via cytoskeleton re-organization. gelsolin is an actin-cytoskeleton regulatory protein that severs or polymerizes actin regulated by local calcium, PIP2 concentration or pH. We previously demonstrated that gelsolin expression was decreased in oral precancer lesions while restored to certain extent in later cancer stages and exhibited prognostic significance. In this study, the role of gelsolin in oral cancer cell invasion, motility and adhesion were evaluated in oral cancer lines through genetic manipulation. Transwell invasion chamber assay and time-lapse video microscopy revealed that gelsolin overexpression did not or only silightly increase cell motility, but significantly enhance cellular invasion into Matrigel. In zymogarphy assay, gelsolin was found to have no significant influence on proteolysis activity of collagen in OEC-M1 cell line. The effect of gelsolin expression on cell aggregation was evaluated by slow and fast aggregation assays. In cell adhesion assay, Cell line over express gelsolin displays stronger adhesion to the collagen coated surface. To further assess the invasion capability of OEC-M1 cells in tissues, embryonic chick heart fragments were served as the model. Increased gelsolin expression was found to promote invasion of OEC-M1 cells into the tissue fragments. Furthermore, the mechanical and topographical properties of the cancer cells were evaluated by Atomic Force Microscopy (AFM). Increased membrane ruffling and pseudopodium formation were observed in gelsolin overexpression OEC-M1 cells. These cells also presented stronger adhesion to the extracellular matrix as greater force was required to move them, which is consistent with our previous results of cell adhesion assay. In summary, gelsolin may participate in oral carcinogenesis progression through enhanced tumor invasion but not enhanced MMP activities. No significant contribution of cell motility enhancement was also noticed. The enhanced cell to extracellular matrix adhesion while decreased cell-cell adhesion may lead to their enhanced invasiveness. The cell to cell and cell to ECM adhesion was further analyzed for their physical properties by atomic force microscopy and consistent results were observed.

Contents
中文摘要.....................................................i
Abstract....................................................iv
誌謝........................................................vi
Contents..................................................viii
Figure contents..............................................x
Introduction.................................................1
1.1 Oral cancer..............................................1
1.2 Invasion and Metastasis..................................2
1.3 Gelsolin.................................................3
1.4 Gelsolin and invasion....................................6
1.5 Atomic force microscope (AFM)............................7
Material and Methods.........................................9
2.1 Reagents.................................................9
2.2 Prepare collagen from rat tail...........................9
2.3 Cell lines and Cell culture.............................10
2.4 Time lapse videomicroscopy analysis.....................10
2.5 Western blot............................................11
2.6 Zymography..............................................11
2.7 Matrigel invasion assay.................................12
2.8 Cell adhesion assay.....................................13
2.9 Cell aggregation assay..................................13
2.10 Chick heart invasion assay.............................14
2.11 Atomic Force Microscopy assay..........................14
Results.....................................................15
3.1 Expression levels of gelsolin in OEC-M1 and other gelsolin transfected stable clone....................................15
3.2 Effect of gelsolin expression on cellular invasion......15
3.3 Effect of gelsolin expression on cellular motility......16
3.4 Effects of gelsolin expression on extracellular protease activity....................................................17
3.5 Gelsolin overexpression inhibits cell aggregation.......17
3.6 Gelsolin overexpression promoted cell adhesion on collagen gel.........................................................18
3.7 Overexpression gelsolin in OEC-M1 cell line promoted cell invasion in chick heart invasion model......................18
3.8 Atomic Force Microscopy.................................19
3.9 Construction of gelsolin expression plasmid.............19
Discussion..................................................21
Reference...................................................26
Figure......................................................33
作者簡歷....................................................45

Figure contents
Figure 1 The gelsolin levels of stabel cell line............33
Figure 2 Matrigel invasion assay............................34
Figure 3 Time-lapse.........................................35
Figure 4 Zymograpy..........................................36
Figure 5 Slow cell aggregation assay........................37
Figure 6 Cell adhesion assay................................38
Figure 7 Chick heart invasion assay.........................39
Figure 8 Atomic Force Microscopy in dry model...............40
Figure 9 Atomic Force Microscopy in liquid model............41
Figure 10 Gelsolin promoter cell invasion model.............42
Figure 11 Construction of gelsolin expression...............43
Figure 12 Construction of gelsolin expression for Fluorescence resonance energy transfer...................................44
                                    

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校內：2006-08-26公開
校外：2007-08-26公開

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