臺灣博碩士論文加值系統

癌症轉移在現今是一個非常熱門的生醫研究課題，而腫瘤細胞(Tumor Cells)在轉移的過程中，經由Epithelial-Mesenchymal Transition (EMT) 的轉換過程，脫離原生腫瘤(Primary Tumor)進入血管，再藉由血管進行遠端轉移。這些在血管中循環流動的癌細胞我們稱為循環腫瘤細胞(Circulating Tumor Cells, CTCs)。目前對於CTCs的研究尚有許多不清楚的地方。CTCs在血液中會受到血流剪切力(shear stress)的刺激，而血流剪切力可能會造成CTCs功能性的改變，包括細胞移動能力、耐剪切力能力(耐沖性)、與細胞附著能力相關的Focal Adhesion 、細胞形變能力等等……。我們使用一個簡易的流體剪切力模擬裝置，讓頭頸癌細胞株(Head and Neck Cancer Squamous Cell Carcinoma, HNSCC)懸浮在液體內，利用剪切力刺激細胞，來回沖15次(兩個小時) ，比較剪切力刺激對兩種不同形態(phenotype)的頭頸癌細胞株(高侵襲性的OECM-1和低侵襲性的FaDu) 所造成的影響。實驗結果顯示，受到10 dyne/cm2剪切力刺激兩個小時後，兩種細胞株的存活率，並沒有顯著的改變；顯示頭頸癌細胞對於10 dyne/cm2的剪切力具有耐沖性。其他進一步實驗結果顯示OECM-1細胞株受到剪切力刺激後，移動能力和Focal Adhesion均明顯的上升，而且具有調降活性氧化物(ROS) 的功能。相對地，FaDu細胞株受到剪切力刺激後，上述各種特性幾乎不受影響。此外我們利用動態影像微粒追蹤微流變學(Video Particle Tracking Microrheology, VPTM)量測細胞的形變能力(彈性)，結果顯示受到剪切力刺激的OECM-1和FaDu兩種細胞株都會變軟(彈性係數下降)。綜合以上所列各項實驗結果，我們認為血流剪切力的刺激，可能有助於高侵襲性的頭頸癌細胞株(OECM-1)的轉移能力。

Cancer metastasis has been a topic of intensive research. In cancer metastasis, tumor cells from primary tumor site undergo Epithelial-Mesenchymal Transition (EMT), enter circulatory system to travel along with the blood stream [in the form of circulating tumor cells (CTCs)] before they metastasize at one or more secondary sites. Fluidic shear stress on CTCs may play an important role in metastasis by changing the properties of CTCs, including resistance to apoptosis, adhesiveness, deformability, etc. In this study, we applied a fluid shear stress within physiological range (~ 10 dyne/cm2) for two hours on two cell lines of head and neck squamous cell carcinoma (HNSCC), OECM-1 (mesenchymal phenotype) & FaDu (epithelial phenotype), to investigate the effects of shear stress on CTCs, including cell viability, cell migration, intracellular stiffness and resistance to reactive oxygen species (ROS). Our results indicated that fluidic shear stress did not affect the viability of both OECM-1 and FaDu cells. It did, however, enhanced the migration capability, focal adhesion, and resistance to ROS to promote metastasis of OECM-1 mesenchymal cancer cells. In contrast, the corresponding effects of fluidic shear stress on FaDu were either much smaller or insignificant. Furthermore, fluidic shear stress reduced the intracellular stiffness of both OECM-1 and FaDu cells. These findings suggest that fluid shear stress may promote metastasis potential of mesenchymal cancer cells.

目錄

摘要 i
Abstract ii
目錄 iii
圖目錄 v
第一章 緒論1
1.1 癌症 (Cancer) 1
1.2 頭頸部鱗狀上皮細胞癌 (Head and Neck Squamous Cell Carcinoma, HNSCC) 2
1.3 癌症轉移 (Cancer Metastasis) 3
1.4 循環腫瘤細胞 (Circulating Tumor Cells, CTC) 4
1.5 血流剪切力 (Hemodynamic Shear Stress) 5
1.6 細胞骨架及細胞遷移 (Cytoskeleton and Cell Migration) 8
1.7 研究動機及目的 (Motivation and Objectives) 10
第二章 實驗材料、架設及方法 12
2.1 細胞培養 (Cell Culture) 12
2.2 剪切力模擬系統 (Fluidic Shear Stress System) 13
2.3 流式細胞技術 (Flow Cytometry, FCM) 15
2.4 細胞隨機遷移試驗 (Cell Random Migration Assay) 16
2.5 細胞刮傷癒合試驗 (Wound Healing Assay) 18
2.6 以螢光標記量化細胞骨架轉接蛋白 (Fluorescence Labeling for the Quantification of Paxillin) 19
2.7 以螢光標定量化活性氧物質 (Fluorescence Labeling for the Quantification of Reactive Oxygen Species, ROS) 21
2.8 動態影像微粒追蹤微流變學 (Video Particle Tracking Microrheology, VPTM ) 23
第三章 實驗結果 25
3.1 HNSCC細胞株受流體剪切力後的細胞存活率 25
3.2 流體剪切力促進OECM-1細胞的移動速率 27
3.2.1 細胞隨機遷移試驗 (Random Migration Assay) 27
3.2.2 細胞刮傷癒合試驗 (Wound Healing Assay) 29
3.3 流體剪切力促進OECM-1內部Paxillin活化 33
3.4 流體剪切力降低OECM-1細胞株的ROS生成 35
3.5 流體剪切力降低OECM-1和FaDu內部彈性 37
第四章 結論與討論 39
第五章 參考文獻 43
第六章 附錄 47

圖目錄

圖1. 血流剪切力示意圖。 6
圖2. 實驗流程圖。 11
圖3. 頭頸癌細胞在顯微鏡20倍明視野下所觀察的細胞型態。 12
圖4. 剪切力模擬系統。 14
圖5. OECM-1細胞在20倍物鏡下的明視野顯微影像和隨機遷移軌跡圖。 17
圖6. 癒合試驗示意圖。 18
圖7. Paxillin螢光染色分析流程圖。 20
圖8. OECM-1內部標定ROS螢光顯微影像。 22
圖9. VPTM流程圖。 24
圖10. 流式細胞儀散射光圖譜。 25
圖11. 細胞存活率百分比圖。 26
圖12. 流體剪切力刺激頭頸癌細胞株提升移動能力。 27
圖13. 細胞移動能力相對變化率。 28
圖14. 細胞刮傷癒合試驗中FaDu顯微影像。 29
圖15. 細胞刮傷癒合試驗顯示流體剪切力不影響FaDu移動能力。 30
圖16. 細胞刮傷癒合試驗中OECM-1顯微影像。 31
圖17. 細胞刮傷癒合試驗顯示流體剪切力促進OECM-1移動能力。 32
圖18. OECM-1染paxillin的螢光顯微影像。 33
圖19. Paxillin面積統計結果。 34
圖20. 螢光標定FaDu內ROS的螢光強度。 35
圖21. 螢光標定OECM-1內ROS的螢光強度。 36
圖22. VPTM量測細胞彈性條件。 37
圖23. 流體剪切力降低細胞內部彈性。 38
圖24. 流體剪切力影響循環腫瘤細胞功能及訊息傳遞的主要路徑。 42
附錄圖a. 有無剪切力刺激之OECM-1細胞移動能力比較。 47
附錄圖b. 有無剪切力刺激之FaDu細胞移動能力比較。 48
附錄圖c. 有無剪切力刺激之MDA-MB-231細胞移動能力比較。 49

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