臺灣博碩士論文加值系統

腫瘤形成的過程中，癌細胞存在體內力學及化學動態變化的微環境。細胞力學在癌症研究扮演重要的角色，因為在上皮細胞間葉化的過程對細胞骨架的影響及細胞力學代表生理的重要意義都須被闡明。細胞力學已經可以表示細胞增殖差異及區分不同生理狀態的細胞，例如：侵襲性強的乳癌、前列腺癌及肺癌細胞有較大的traction stress。在2008年，Torng, Lee等人利用microarray 分析人類卵巢子宫内膜癌細胞 (OVTW59-P0) 及其侵襲性相關亞型細胞 (P1-P4，依序其侵襲性增加) ，發現其中一組基因和癌症侵襲能力呈負相關，但尚未有研究證實人類卵巢子宫内膜癌細胞及其侵襲性相關亞型細胞在細胞力學的差異。
在本研究中，我們利用微板量測系統 (MMS) 的方法量測人類卵巢子宫内膜癌細胞 (OVTW59-P0) 及其侵襲性相關亞型細胞 (P4) 的細胞力學，這套系統是利用具彈性的微探針去測量細胞勁度 (Pa) 和nN解析度下單顆細胞的貼附力 (nN) 。利用MLCK抑制劑 (ML-7) 及actin破壞劑 (Cyto-D) 使細胞骨架動態改變並量測其細胞力學，並用細胞增殖能力、群體細胞爬行、細胞穿越、及單顆細胞爬行等分析來比較P0與P4間的功能性差異。
我們的研究結果顯示人類卵巢子宫内膜癌細胞侵襲性增加，細胞變軟 (較P0減少30%) 但貼附力並無顯著差異。高侵襲性亞型細胞 (P4) 其勁度及貼附力受MLCK抑制劑 (ML-7) 與actin破壞劑 (Cyto-D) 也有顯著影響 (較P0減少70%)，從細胞行為分析， P4的單顆細胞及群體二維位移爬行分別比P0快30%及80%，三維穿越能力較P0強17%，在ML-7及Cyto-D抑制後細胞勁度變軟，P0和P4原本的細胞行為差異也跟著消失。
總結，細胞力學可以明顯區分P0和P4的差異。細胞骨架重組後，我們證明高侵襲性EC細胞P4在藥物抑制後，因為沒有適當的細胞骨架支撐，細胞勁度變得更軟，其移動能力也變弱。這也指出當癌細胞轉移時須透過細胞骨架調整才能維持適宜的細胞勁度以利於細胞移動。因此微板量測系統成功區辨了OVTW59-P0及P4的細胞力學差異以及P4加藥抑制後的細胞勁度變軟其移動能力也會降低，因此，可以使用微板量測系統量測細胞力學並且區分生理行為的細胞，這對癌症的臨床診斷及治療的應用也是很重要的幫助。

Cancer cells exist in a mechanically and chemically heterogeneous microenvironment which undergoes dynamic changes throughout neoplastic progression. The cell mechanical properties (CMs) have been shown to differentiate the proliferation and differentiation of cells in different physiology states. The importance of CMs to cancer is appreciated, yet the contributions of specific cytoskeleton to cells undergoing malignant transformation and their physiological significance need be further elucidated. In human metastatic breast, prostate and lung cancer cell lines, traction stresses were significantly increased compared to non-metastatic counterparts using traction force microscopy. By microarray analysis, the human ovarian endometrioid carcinoma (EC) cell line OVTW59-P0 and its invasion-related sublines (P1-P4, in the order of increasing invasive activity) have been found that one group of gene was negatively correlated with cancer invasiveness .However, the identified different CMs among the invasive-related sublines (P0 and P4) remain unknown.
In this study, a microplate measurement system (MMS) approach based on the deflection of a flexible micro-cantilever was utilized to measure the stiffness (in Pa) and adhesion force (in nN) of a single cell (P0 and P4) with nN resolution. And the effects of cytoskeletal dynamics in the difference of CMs were examined by MLCK inhibitor (ML-7) and actin disrupter (Cyto-D). To compare the functional difference among P0 and P4, we executed cell proliferation assays, collective migration, invasion assay, and single cell migration analysis.
Our results demonstrated that in line with increased invasiveness, so-called P4 cells, and the EC cells became softer by 30% but remained unchanged in adherence. Stiffness and adherence of P4 cells were dramatically affected by ML-7 and Cyto-D, especially in tensile stiffness (decreased by 70%). Functionally, the single or collective motility and invasiveness of P4 was superior to P0 by 30%, 80%, and 17%, respectively. In line with the decreased CMs, the differences between P0 and P4 in mobility were eliminated by ML-7 and Cyto-D inhibition.
In conclusion, the differences between P0 and P4 could be differentiated well by cell mechanical properties. From the cytoskeletal aspect, this study demonstrated that the highly invasive EC cell, P4, after drug inhibition became much softer and lower motility without appropriate actomyosin accommodation. It indicated that cancer cells needed the adapted cytoskeletal dynamically to maintain the appropriate stiffness to invade. Therefore, the use of this MMS to measure mechanical properties of cells and differentiate physiological state was available. There will be a significant help and applicability for clinical diagnosis and treatment of cancer.

ABSTRACT I
摘要 III
致謝 V
TABLE OF CONTENTS VI
LIST OF TABLES VIII
LIST OF FIGURES IX
CHAPTER 1 INTRODUCTION 1
CHAPTER 2 METHODOLOGY 3
2.1 Materials and Instruments 3
2.2 Cell culture 4
2.3 Immunofluorescence staining 5
2.4 Cell proliferation assays 5
2.5 Cell mechanical properties using microplate measurement system 7
2.6 Wound healing assay 10
2.7 Cell invasion assay 11
2.8 Time-lapse cell migration analysis 13
2.9 Image analysis 13
2.10 Statistical analysis 13
CHAPTER 3 RESULTS 15
3.1 Analysis of the influence of ML-7 and Cyto-D on OVTW59 cell line proliferation in vitro 15
3.2 Effects of ML-7 and Cyto-D on F-actin and pMLC 17
3.3 Cell mechanical properties alteration and addition of inhibitor 17
3.4 Cell motility 22
CHAPTER 4 DISCUSSION 28
4.1 The cell proliferation difference of P0 and P4 28
4.2 The CMs difference of P0 and P4 28
4.3 The effect of ML-7and Cyto-D 30
4.3.1 The effect of F-actin and pMLC after ML-7 and Cyto-D inhibition 31
4.3.2The effect of cell proliferation after ML-7 and Cyto-D inhibition 31
4.3.3The effect of CMs after ML-7 and Cyto-D inhibition 31
4.3.4The effect of single cell after ML-7 and Cyto-D inhibition 32
4.4 Summary 32
4.5 Limitation 33
CHAPTER 5 CONCLUSION 34
REFERENCES 35

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