臺灣博碩士論文加值系統

大腸結直腸癌是目前全球癌症死亡的重要原因，儘管現在已經有標靶藥物改善病患的預後，但仍在世界癌症相關死亡主因排行中高居第二，顯示目前的藥物療效仍然有限。自噬作用是細胞內新陳代謝的路徑，在最近幾年有很多研究指出其和癌症有相關性，其中，ATG7 是自噬核心的調控基因，它的作用也類似於泛素樣蛋白的E-1 enzyme，會透過和泛素樣蛋白結合來幫助它們向E-2 enzyme轉移，因此ATG7在泛素系統當中做為自噬體初始的重要調節因子。目前已有其它研究報導ATG7在其他癌症中高表達會促進自噬作用，但在大腸結直腸癌當中還沒有太多研究，因此設計了三條反義寡核苷酸 (ASO) 來抑制ATG7的表現，以了解自噬對大腸結直腸癌細胞的影響。根據我們的結果顯示，使用ATG7-ASO處理細胞72小時後，ATG7的蛋白質表現及細胞的存活率明顯的被抑制。同時，透過流式細胞儀檢測發現，凋亡細胞有增加，而細胞週期有受到影響，凋亡蛋白酶3 (caspase-3) 活性及其下游分子聚腺苷酸二磷酸核糖基聚合酶 (Poly ADP-ribose Polymerase，PARP) 的剪切型態都明顯增加，通過添加細胞死亡抑制劑，添加了凋亡抑制劑之後，細胞的存活率會回升，因此，推論ATG7-ASO會導致細胞凋亡。此外， ATG7-ASO會影響細胞的活性，也會抑制腫瘤球體的形成與生長活性，因此， ATG7-ASO在未來或許可以作為治療大腸結直腸癌的潛在治療藥物。

Colorectal cancer currently ranks as a significant cause of cancer-related deaths worldwide, despite advancements in treatments such as surgery and chemotherapy. It remains the second most common cause of cancer-related deaths globally. In recent years, there has been increasing research into the relationship between autophagy and cancer. ATG7, an autophagy-related regulatory gene, functions similarly to the E-1 enzyme in the ubiquitin-like protein system. It assists in the transfer of ubiquitin-like proteins to the E-2 enzyme, thus serving as an initiator and crucial regulatory factor in autophagosome asSDbly within the ubiquitin system. While studies have reported high expression of ATG7 promoting autophagy in other cancers, its role in colorectal cancer remains relatively underexplored. Therefore, three antisense oligonucleotides (ASO) were designed to inhibit the expression of ATG7. To understand the impact of autophagy on colorectal cancer cells. According to our results, after treating cells with ATG7-ASO for 72 hours, the protein expression of ATG7 and the survival rate of cells were significantly inhibited. Moreover, it was found that apoptotic cells was increased and the cell cycle was impacted through flow cytometry. The activity of apoptotic protease (caspase-3) and the cleavage form of its downstream molecule poly ADP-ribose Polymerase (PARP) are significantly increased, and cell death is inhibited by adding an apoptosis inhibitor, the cell survival rate will increase. Therefore, it is inferred that ATG7-ASO will cause cell apoptosis. In addition, ATG7-ASO can inhibit the formation and activity of tumor spheroids and. Therefore, ATG7-ASO may be used as a treatment in the future. Potential therapeutic drugs for colorectal cancer.

論文審定書 ⅰ
中文摘要 ⅱ
Abstract ⅲ
目錄 ⅳ
圖次 ⅶ
縮寫表 ⅷ
第一章 背景介紹1
1.1 大腸結直腸癌 (Colorectal cancer, CRC) 1
1.2 細胞自噬 (Autophagy)2
1.3 自噬在癌症中的作用5
1.4 自噬因子ATG7 (Autophagy related 7) 在自噬過程中參與的作用5
1.5ATG7在癌症中的作用6
1.6反義寡核苷酸 (Antisense Oligonucleotide, ASO) 的分子機制及用途7
1.7研究動機及目的9
第二章 材料與方法10
2.1 化學藥品10
2.2 細胞培養 (Cell culture)12
2.3 ASO轉染 (ASO transfection)15
2.4 西方墨點法 (Western blot)16
2.5 細胞存活率測試 (Cell viability assay)25
2.6 細胞週期分析 (Cell cycle phases analysis)26
2.7 癌細胞球體形成試驗 (Sphere formation assay) 29
2.8 活/死細胞螢光試驗 (Live/Dead cytotoxicity assay)28
2.9 球體細胞存活率試驗 (3D sphere cell viability assay)30
2.10 活性氧化物質產生試驗 (Reactive oxygen species assay)30
2.11 粒線體膜電位分析 (Mitochondria membrane potential assay) 31
2.12 蛋白質體學分析 (Proteomics analysis)32
第三章 實驗結果34
3.1 ATG7-ASO對CRC細胞的蛋白質表現的影響34
3.2 ATG7-ASO對CRC細胞存活率的影響35
3.3 ATG7-ASO對細胞週期的影響35
3.4 ATG7-ASO對CRC細胞中Caspase-3/7的活性測定36
3.5 細胞死亡抑制劑對CRC細胞的存活率影響36
3.6 CRC細胞中p53的參與狀況37
3.7 CRC細胞中活性氧化物質的參與狀況38
3.8 CRC細胞中粒線體膜電位分析38
3.9 ATG7-ASO對CRC細胞的球體形成能力影響39
3.10 ATG7-ASO對HCT116細胞的蛋白質體分析39
第四章 討論42
第五章 結論46
第六章 實驗結果圖47
參考文獻59

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