根據臨床報告，肝硬化程度對於肝癌細胞的發展有一定程度的影響，但詳細機制仍尚未明確。在本研究中，我們探討肌動蛋白變異的在不同基質硬度微環境下，對於肝癌細胞的生理特性與機械訊息傳導影響中所造成的影響。實驗中，以人類肝癌Alexander cell(PLC/PRF/5 ATCC® CRL-8024) 和過度表達 Kappa-actin的Alexander cell(Alex-)來做為研究模型，並使用可調控硬度的聚丙烯醯胺基質來模擬人體肝臟硬度環境。
首先，觀察到硬度能夠改變細胞的形態大小，接著更進一步探討細胞與外細胞基質間的連接focal adhesion (FA)，發現硬度及肌動蛋白變異影響FA的形成與細胞牽引力(traction force)的大小，進而造成肝癌細胞的黏附能力也隨之改變。而細胞的黏附於不同環境時也會影響細胞的增生能力，細胞在硬度高的環境下增生情形較明顯，其可能原因與focal adhesion kinase(FAK)有關聯性。考量FA對於肝癌細胞轉移的影響，在觀察單顆細胞的遷移中，發現低硬度環境及肌動蛋白變異使得細胞形成較少FA及較低的細胞牽引力，促使細胞有較快的移動速率。而在群體細胞在硬度高的環境下移動速率快且有一致的方向性，推測與細胞之間的adherens junction(AJ)調節有關。最後探討肝癌細胞的異位轉移，即觀察肝癌細胞的在三維空間中侵入情形，肌動蛋白變異使細胞侵入能力降低。
從本研究得知，外部環境硬度增加如同肝硬化程度上升，肝癌細胞的生理特性也隨之改變。細胞內肌動蛋白的變異會改變細胞內FA與AJ 的生成，進而影響肝癌細胞的黏附、增生、遷移與侵入。

Liver cancer is one of the most common cancer in the world, which is highly associated with liver cirrhosis. Thought stiffer extracellular matrix is speculated to facilitate the development of hepatocellular carcinoma development, the detail mechanisms remain to be elucidated. In this study, we investigated the interconnections of substrate stiffness and actin mutation on the HCC cellular physiology.
HCC cell line Alexander (PLC/PRF/5 ATCC® CRL-8024) and Alex- (cells overexpressing Kappa-actin) were selected as study models, and the stiffness tunable polyacrylamide matrixes were applied to mimic the cirrhotic microenvironments. We noticed the spread area of HCC cells increased on stiffer substrates, which consisted with the qPCR data of focal adhesion (FA). In addition to ECM-cell anchorage, in Alex-, the alteration of FA leads to different cell-ECM dynamic interactions. The unique -actin downregulated the formations of FA and adherens junction at the cell-cell contact. The changes of junction formation may contribute to the variety of cell migration on different ECM rigidity. Moreover, the data of traction force microscopy indicated that the mechanical force reduction leaded by actin mutant, which may related to the less invasion observed in our in vitro experiments.
In summary, our results demonstrated that the changes of cell-cell/ cell/ECM adhesion of HCC cell in response to the ECM stiffness, and the expression of -actin can modify the mechano-responses of HCC through cell adhesion alterations.

中文摘要 (II)
Abstract (III)
致謝 (IV)
章節目錄 (V)
圖目錄 (VIII)
表目錄 (X)
第一章 序論 P.1
1-1肝癌 P.1
1-2肝硬化 P.1
1-3物理微環境對癌症發生的影響 P.2
1-5細胞與外細胞基質的細胞牽引力(traction force) P.9
1-6實驗動機 P.11
第二章 材料與方法 P.12
2-1細胞培養 P.12
2-2 聚丙烯酰胺膠(Polyacrylamide gel) P.12
2-3免疫螢光染色 P.15
2-4 Q-PCR(Quantitative real time polymerase chain reaction)P.16
2-5黏附分析(Adhesion assay) P.17
2-6增殖分析(Growth assay) P.17
2-8細胞遷移(migration) P.18
2-8三維細胞培養 P.18
2-9蛋白膠(collagen gel)與侵入(invasion) P.18
2-10細胞牽引力顯微鏡(traction force microscope) P.18
2-11資料統計整理方法 P.19
2-12實驗材料與儀器 P.19
第三章 結果與討論 P.22
3-1細胞在不同硬度上形狀與大小 P.22
3-2過度表達-actin改變肝癌細胞的FA P.25
3-3不同硬度基質上的黏附力 P.29
3-4不同硬度基質上的增生率 P.29
3-5 肝癌細胞的遷移 P.31
3-6 細胞的侵入 P.37
第四章 結論 P.40
參考文獻 P.41

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