細胞和細胞外間質（Extracellular matrix, ECM）的連接對細胞的存活與分化有極大的影響，當一般的細胞移動到新的環境時無法存活，有一部分原因是因為人體中的各個組織的ECM組成成分有所不同，然而，癌細胞具有適應新環境的能力，我們的目標是探討癌細胞如何改變周遭微環境，並從中生存下去? ECM的組成成分與基因在現有的論文中少有提及。我們參考了掃描人體及小鼠的全基因去預測人類及小鼠的ECM基因的論文，現有的技術，針對蛋白質產物都有其極限，於是我們嘗試開發使用新的工具，以即時定量聚合酶連鎖反應（real-time quantitative polymerase chain reaction, RT- qPCR）的技術為基礎，自行設計引子並將其優化到最佳條件，確保新工具的準確性及效率性，並將其用於大量樣品的掃描測試。根據我們實驗室之前的研究得知，細胞會根據環境改變ECM基因的表現量。因此我們使用懸浮培養環境系統化來掃描人類ECM基因的調控，並依據結果將基因分組，來探討各組的調控機制。基因表現會受轉錄因子調控。我們發現特定轉錄因子的過度表現能抵抗失巢凋亡（anoikis）。接著，我們利用自行開發的工具來探討特定轉錄因子對ECM基因表現的控制。

Cell survival is dependent on their connection with ECM. The composition of ECM in each part of the human tissue varies. Normal cells cannot survive when moving to new environments. It highlights the importance of how metastatic cancer cells gain the ability to survive in the new environments? The composition and genes of ECM are rarely mentioned in the existing papers, and we refer to the papers which predictive whole-gene scanning from human and mouse. The existing technologies, that help to investigate proteins expression, are not suitable for this study because the dependence of antibody. We developed a new tool based on the RT-qPCR (real-time quantitative polymerase chain reaction) for screening a large number of tests. The papers published in our laboratory before found that the cells will change ECM genes performance basis on difference of environments. Therefore, we used suspension culture to systematically scan human ECM genes. The result could be used to divide ECM genes into groups and to explore the regulatory mechanisms of human ECM genes. Gene expression is controlled by transcription factors. We found that the cell line which overexpression of specific transcription factors can resistant to anoikis. Next, we used our tools to explore the regulate of ECM gene expression by specific transcription factors.

目錄
中文摘要………………………………………………………………………………2
Abstract……………………………………………………………………………….3
致謝……………………………………………………………………………………4
第一章 介紹 …………………………………………………………………………7
1.1 癌症……………………………………………………………………………….7
1.2 癌症轉移………………………………………………………………………….7
1.3 乳癌……………………………………………………………………………….7
1.4 細胞的貼附性…………………………………………………………………….8
1.5 細胞外間質……………………………………………………………………….8
1.5.1 Extracellular matrix (ECM)………………………………………………...8
1.5.2 Integrin……………………………………………………………………..9
1.6 轉錄……………………………………………………………………………….9
1.6.1 轉錄因子…………………………………………………………………10
1.6.2 JUND……………………………………………………………………...10
1.7生物微環境………………………………………………………………………10
1.8 ECM對癌症的重要性…………………………………………………………..10
1.9 檢測方法………………………………………………………………………...11
1.9.1 immunohistochemistry (IHC)…………………………………………….11
1.9.2 Western blot………………………………………………………………11
1.9.3 Quantitative Real-Time polymerase chain reaction (qPCR)……………..12
第二章 實驗結果……………………………………………………………………13
2.1 ECM的基因預測………………………………………………………………..13
2.2 RT-qPCR引子……………………………………………………………………13
2.2.1 引子設計…………………………………………………………………13
2.2.2 引子改良…………………………………………………………………14
2.3 懸浮細胞………………………………………………………………………...14
2.3.1 懸浮細胞培養……………………………………………………………14
2.3.2 懸浮細胞ECM基因表現……………………………………………….15
2.3.3 轉錄因子預測……………………………………………………………17
2.4 過量表現轉錄因子的細胞……………………………………………………...18
2.4.1 懸浮培養過量表現JUND的細胞………………………………………18
2.4.2 懸浮細胞過量表現JUND後ECM基因表現………………………….18
第三章 討論…………………………………………………………………………20
第四章 材料與方法…………………………………………………………………33
第五章 參考資料……………………………………………………………………35
補充資料……………………………………………………………………………..38

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