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系統識別號 U0026-1908201315105500
論文名稱(中文) 經微板系統量測上皮細胞間質化
論文名稱(英文) Nanomechanical Analysis of Epithelial to Mesenchymal Transition by Microplate Mechanical Measurement System
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 101
學期 2
出版年 102
研究生(中文) 胡瓊文
研究生(英文) Chiung-Wen Hu
學號 p86001192
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 指導教授-葉明龍
口試委員-艾群
口試委員-鐘震桂
口試委員-邱文泰
中文關鍵字 上皮細胞間質化  轉化生長因子β1  細胞勁度  肺癌 
英文關鍵字 Epithelial to mesenchymal transitions (EMT)  Transforming growth factor-β1 (TGF-β1)  Cell stiffness  Lung cancer 
學科別分類
中文摘要 上皮細胞 - 間質化(EMT),意指上皮特定形態的損失,發生在多種生理活動,包括傷口癒合,器官纖維化,並開始轉移的癌症進展。在體外TGF-β1能夠成功地誘導EMT。與EMT相關的細胞力學並未有深入的研究。本研究將藉由TGF-β1誘導EMT,使用正常小鼠乳腺上皮細胞(NMuMG),人類肺腺癌細胞(A549)和小鼠肺癌細胞(LLC)在體外進行微孔板機械測量系統(MMS)量測細胞的壓縮和拉伸勁度,使用transwell進行浸潤測試。免疫熒光染色觀察E-cadherin,F-actin肌動蛋白,和vinculin,以驗證形態上和分子交互作用在EMT後表現。我們發現細胞的壓縮勁度、拉伸勁度和粘附力,大多數組別在EMT後顯著增加。在EMT後,所有細胞侵襲能力增加。根據免疫螢光染色,在EMT後F-actin、E-cadherin和vinculin細胞的分佈也改變。實驗提出證實MMS根據它們的機械性能具有區辨EMT能力,無論在正常細胞或癌細胞。它可以提供新的策略,以開發新的方法來診斷或鑑別病變細胞。此外, EMT後的侵襲能力不同,可以藉此來作疾病預防或病程評估。此細胞力學為基礎的研究,可以進一步在未來開發適用於藥物設計,醫用植入體,組織工程,人工神經網絡等領域。
英文摘要 Epithelial-to-mesenchymal transition (EMT), a term describing the loss of epithelium-specific function, occur in several physiological activities including wound healing, organ fibrosis and initiation of metastasis for cancer progression. TGF-1 can successfully induce EMT in vitro. How the cell mechanics associated with EMT has not been well investigated yet. This study investigates the cell compressive and tensile stiffness by microplate mechanical measurement system (MMS) and invasion by transwell in vitro of normal murine mammary gland epithelium (NMuMG), adenocarcinomic human alveolar basal epithelial cells (A549), and Lewis lung carcinoma (LLc) associated with EMT under TGF-1 induction. Immunofluorescence staining was used to observe intercellular connection molecule (E-cadherin), the cytoskeleton (F-actin), and focal adhesion molecule (vinculin) to verify the induction of EMT and morphology and molecular alternation after EMT. The compressive and tensile stiffness and adhesion force of the studied cells increase significantly after EMT in most of groups. The invasion ability of all studied cells also increases after EMT. According to immunofluorescence staining, the arrangement of F-actin and the distribution of E-cadherin and vinculin of the studied cells also change after EMT. The present experiment confirmed MMS has the ability to differentiate EMT according to their mechanical properties for both in normal cells or cancer cells. It can provide new stratagem to develop novel approach to diagnose or distinguish diseased cells. Furthermore, the different ability of invasion after EMT can be used to separate the migration and metastasis of the cancer cells for disease prevention or progression. This cell mechanics based study can be further developed to apply in drug design, medical implants, tissue engineering, artificial neural networks and other areas in the future.
論文目次 Table of Contents
摘要 i
Abstract ii
致謝 iv
Chapter 1 Introduction 1
1.1 Cell Mechanics 1
1.1.1 Mechanical properties of the cell 2
1.2 Epithelial-mesenchymal transition (EMT) 6
1.3 Cancer 11
1.3.1Lung cancer 12
1.3.2Diagnosis of lung cancer 15
1.3.3Treatment of Lung Cancer 17
1.4 Motivation 18
1.5 Purposes 19
Chapter 2 Methodology 20
2.1 The flow chart of this study is described in the following figure 20
2.2 Cell line 23
2.2.1 Harvest of NMuMG 23
2.2.2 Harvest of A549 23
2.2.3 Harvest of LLc 24
2.3 TGF-β1 induced EMT 24
2.4 Coating ECM (Type I collagen) 24
2.5 Prepare cell for Microplate mechanical measurement system (MMS) 25
2.6 Experimental of microplate mechanical measurement system (MMS) 25
2.7 Biomechanical measurements 26
2.8 Immunofluorescent Staining 29
2.9 Invasion assays 30
2.10 Analysis and Statistics 31
Chapter 3 Results 32
3.1 Mechanical properties measurement 32
3.2 Immunofluorescent Staining of NMuMG 37
3.3 Immunofluorescent Staining of A549 38
3.4 Immunofluorescent Staining of LLc 40
3.5 Invasion assay 42
Chapter 4 Discussions 44
4.1 Immunofluorescent staining 44
4.2 Cancer cell invasion ability after EMT 45
4.3 Mechanical properties of cells under EMT 46
4.4 MMS in mechanics measurements 48
Chapter 5 Conclusions 50
5.1 Future work 50
References 51
Appendix 59
Animal model 59
Tumor growth trend 59
Pathological 61
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