中文摘要
　　肝癌在世界上為第五大常見癌症以及第三大致死率癌症。許多致癌風險因子如B型與C型肝炎病毒或是病患有長期酗酒行為，都會引發慢性發炎，造成細胞外基質過度累積在肝臟形成肝硬化，進而演變成肝癌Hepatocellular carcinoma (HCC)。細胞外基質與細胞間機械訊號都會改變細胞的生理型態，包括生長、轉移以及入侵等。其中癌細胞與細胞外基質間的細胞機械力與癌病惡化轉移有高度相關，因此我們透過牽引力顯微鏡量測肝癌細胞在不同微環境下的機械訊號變化。
　　本研究中，以人類肝癌Alexander cells (PLC/PRF/5 ATCC® CRL-8024) 以及過度表達kappa-actin的Alexander cells (Alex-к) 作為實驗模型，藉由影像處理方式來量化細胞在基材上所產生的細胞牽引力大小。Kappa-actin 是一種與術後致死率有高度相關的肌動蛋白突變。實驗發現，當微環境硬度較高時癌細胞產生較大牽引力，有較高的vinculin表現量，顯示focal adhesion表達量與細胞牽引力成正比關係。實驗結果指出Alex會比Alex-к有更大的牽引力，且細胞內肌動蛋白有較高的自組裝效率，推測其細胞在遷移速率較快與牽引力較大的情況下利於癌細胞入侵周遭組織。研究中也發現藉由改變基質硬度或是不同細胞外基質分子會對細胞牽引力造成影響，再而間接調節細胞間張力。
　　從本研究中建立一套細胞牽引力顯微鏡系統，並觀察到當癌細胞感受不同細胞外基質硬度時，其細胞上機械力訊號也隨之改變，而癌細胞中肌動蛋白的突變，也會造成機械力訊號上的變化，進而影響癌細胞黏附、侵入、轉移等生理特性。癌症在轉移過程中，癌細胞型態以及機械力會因微環境不同而有所變化，透過牽引力顯微鏡系統觀測癌細胞生理特性改變，能夠更進一步確認癌症轉移過程，對於後續癌症治療無疑是一大利器。

Abstract
Liver cancer is the fifth most common cancer in the world and the third most common cause of cancer mortality. During liver cancer progression, liver cirrhosis, the abnormal extracellular matrix accumulated in liver, is highly associated with the development of hepatocellular carcinoma (HCC). Numerous studies identified the important gene pathways related to HCC progression, however, the influences of physical microenvironment have not been fully explored yet.
In this study, we established the traction force microscopy to investigate the mechanical responses of HCC in engineered cirrhosis environment. The Alexander cells (PLC / PRF / 5 ATCC® CRL-8024) and cells overexpressing kappa-actin, which is correlated to poor post-operate survival rate, are selected as our cell models. The outcomes of traction force microscopy demonstrated that the traction force of HCC increases with the increasing substrate stiffness, and the expression of focal adhesion increases accordingly. We also observed the alteration of traction force and migration speed of HCC with actin mutant. From our intercellular force analysis, a unique correlation between traction force and intercellular force seems be regulated by actin assembly in HCC.
The results of this study demonstrated the rigidity of microenvironment can alter the mechanical responses of HCC, which is associated with tumor invasion and metastasis. Moreover, the interconnection between cell-ECM traction force and intercellular force through action cytoskeleton may contribute to our current understanding about the transmission of mechanical signals over multicellular structures. In cancer metastasis, variation of microenvironment will change morphology and mechanical force of cancer cells. By traction force microscope, cancer metastasis process can confirm through observed the physiological characteristics changes of cancer cells. Traction force microscope will be a great method to involve cancer treatment in the future.

章節目錄
中文摘要 i
Abstract ii
章節目錄 iii
表目錄 viii
第1章 序論 1
1-1 肝癌與肝硬化 1
1-2 物理微環境對癌症發生的影響 2
1-3 細胞機械力改變細胞生理型態 3
1-4 細胞機械訊號傳遞 8
1-5 牽引力顯微鏡 (Traction force microscopy,TFM) 11
1-5-1 牽引力顯微鏡回顧 12
1-5-2 聚丙烯酰胺膠 13
1-5-3 圖像速度測定技術 15
1-5-4 牽引力場重建方法 16
1-5-5 牽引力顯微鏡的特性與其他量測機械訊號的系統 17


第2章 材料與方法 20
2-1 細胞培養 20
2-2 聚丙烯酰胺成膠 20
2-2-1 玻璃表面活化 20
2-2-3 膠原蛋白與纖維連接蛋白塗佈上聚丙烯酰胺膠 22
2-2-4 聚丙烯酰胺膠滅菌與細胞貼附 22
2-3 藉由旋轉離心使聚丙烯酰胺成膠 24
2-4 牽引力顯微鏡 25
2-4-1 活細胞影像 25
2-4-2 聚丙烯酰胺膠型變 26
2-4-3 迭代互相關函數粒子成像測速法 (Particle Image Velocimetry,PIV) 27
2-4-4 牽引力場重建 28
2-4-5 細胞對張力量測 30
2-5 細胞遷移 31
2-6 免疫螢光染色 31
2-7 Q-PCR (Quantitative real time polymerase chain reaction) 31
2-7-1 抽取mRNA 31
2-7-2 反轉錄反應 32
2-7-3 Q-PCR即時定量 32
2-8 Actin光漂白螢光恢復術 33
第3章 結果與討論 36
3-1 選擇最佳interrogation window大小 36
3-2 單顆細胞的牽引力分析 39
3-4 藉由旋轉離心方法改善螢光影像背景訊號的干擾 44
3-5 肝癌細胞在不同硬度下牽引力分布與表現 47
3-6 細胞貼附面積與總牽引力及細胞單位面積牽引力關係 52
3-7 細胞面積與遷移速率 54
3-8 不同細胞外基質的牽引力 57
3-9 細胞對牽引力分析 61
3-10 改變細胞外基質硬度或生化組成以調節細胞與細胞間張力表現 62
3-11 Latrunculin-B阻止細胞內G-actin聚合 64
第4章 結論 69
參考文獻 71

圖目錄
圖 1 1 肝癌病發途徑 1
圖 1 2 細胞與細胞外基質相互作用產生牽引力 3
圖 1 3 肌肉成纖維細胞產生機械力促使傷口修復 4
圖 1 4 Myosin透過ATP黏附上actin產生細胞收縮力 5
圖 1 5 癌細胞轉移過程 6
圖 1 6 ECM分子與ECM受體訊息傳遞影響癌細胞侵入 7
圖 1 7 細胞內focal adhesion的分子結構 8
圖 1 8 FAK傳遞至下游訊傳蛋白影響細胞遷移與癌細胞入侵 9
圖 1 9 單體G-actin聚合成F-actin 11
圖 1 10 Actin聚合及解聚產生牽引力驅使細胞遷移 11
圖 1 11 細胞種植於垂直的彈性支柱上 12
圖 1 12 纖維母細胞種植於矽膠薄膜上產生皺摺 12
圖 1 13 牽引力顯微鏡系統 13
圖 1 14 丙烯酰胺與BIS的vinyl加成聚合反應 14
圖 1 15 粒子追踪測速技術 15
圖 1 16 粒子影像測速技術 16
圖 1 17 單位interrogation window中所包含粒子個數對於平均位移與平均力誤差值 17
圖 2 1 聚丙烯酰胺成膠製作流程 23
圖 2 2 Sulfo-SANPAH分別與聚丙烯酰胺及I型膠原蛋白進行交聯 23
圖 2 3 聚二甲基矽氧烷夾具 24
圖 2 4 不鏽鋼夾具 25
圖 2 5 細胞產生牽引力造成聚丙烯酰胺膠中奈米螢光珠位移 26
圖 2 6 互相關函數做圖表示與50% interrogation window部分重疊 27
圖 2 7 迭代互相關函數粒子成像測速法操作流程 28
圖 2 8 運用FTTC重建牽引力場 28
圖 2 9 逆問題正歸化算式 29
圖 2 10 細胞對中牽引力與張力方向向量 30
圖 2 11 細胞對牽引力與張力相互作用 30
圖 3 1 不同interrogation window大小內所包含螢光珠數目(N=10) 36
圖 3 2 聚丙烯酰胺膠最上層奈米螢光珠影像 36
圖 3 3 不同interrogation window大小經由PIV計算位移方向向量的解析度 37
圖 3 4 單顆細胞牽引力方向向量與熱點圖 39
圖 3 5 HCC中vinculin聚集情形 40
圖 3 6 HCC貼附在交聯膠原蛋白的6.2kPa聚丙烯酰胺膠下牽引力點分佈 41
圖 3 7 Alex與Alex-κ貼附交聯膠原蛋白6.2 kPa聚丙烯酰胺膠下平均牽引力 42
圖 3 8 HCC在glass組的團簇表現數量 43
圖 3 9 比較新式與舊式的聚丙烯酰胺膠最上層螢光影像與螢光強度 44
圖 3 10 聚丙烯酰胺膠Z軸切面圖 45
圖 3 11 HCC貼附舊式交聯膠原蛋白6.2kPa聚丙烯酰胺膠下牽引力點分布 45
圖 3 12 比較Alex與Alex-κ貼附新式與舊式聚丙烯酰胺膠平均牽引力改變 46
圖 3 13 HCC貼附交聯膠原蛋白1.4 kPa聚丙烯酰胺膠下牽引力點分布 47
圖 3 14 比較Alex與Alex-κ貼附1.4 kPa聚丙烯酰胺膠下平均牽引力大小 48
圖 3 15 比較Alex與Alex-κ貼附不同硬度聚丙烯酰胺膠下平均牽引力大小 48
圖 3 16 肝癌細胞在不同硬度下的vinculin表達量 49
圖 3 17 不同硬度下β1 integrin與Cav1之間的相互調節 50
圖 3 18 Alex與Alex-κ在6.2kPa聚丙烯酰胺膠下總牽引力與細胞單位面積牽引力及細胞貼附面積關係 52
圖 3 19 Alex與Alex-κ在1.4kPa聚丙烯酰胺膠下總牽引力與細胞單位面積牽引力及細胞貼附面積關係 53
圖 3 20 單顆肝癌細胞貼附面積與移動速率關係 54
圖 3 21 單顆肝癌細胞貼附交聯膠原蛋白6.2kPa聚丙烯酰胺膠上移動軌跡 55
圖 3 22 肝癌細胞經光漂白螢光回復系統後actin的回覆時間 57
圖 3 23 HCC貼附交聯纖維連接蛋白6.2 kPa聚丙烯酰胺膠下牽引力點分布 58
圖 3 24 比較Alex與Alex-κ貼附交聯纖維連接蛋白6.2 kPa聚丙烯酰胺膠下 58
圖 3 25 比較Alex與Alex-κ在不同細胞外基質下平均牽引力差異 59
圖 3 26 膠原蛋白與纖維連接蛋白透過不同integrin黏附於細胞外基質上 60
圖 3 27 細胞對牽引力方向向量與熱點圖 61
圖 3 28 Alex細胞間張力和細胞牽引力 62
圖 3 29 Alex-κ細胞間張力和細胞牽引力 62
圖 3 30 鈣粘蛋白與牽引力不平衡調節EMT 64
圖 3 31 Latrunculin-B阻止G-actin聚合影響HCC型態與牽引力 65
圖 3 32 Latrunculin-A or B黏附使G-actin無法聚合成F-actin 65
圖 3 33 HCC加入Latrunculin-B 6分鐘後牽引力點改變 67
圖 3 34 比較Alex與Alex-κ在加入Latrunculin-B 6分鐘後平均牽引力差異 68

表目錄
表格 1 1 涉及癌細胞入侵的細胞外分子 7
表格 1 2 BEM與FTTC優缺點比較 17
表格 2 1 不同硬度下丙烯酰胺膠成分比例 22
表格 2 2 PCR使用之引子 32
表格 2 3 實驗材料與儀器 34

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