臺灣博碩士論文加值系統

研究指出惡性腫瘤為一高度異質性的組織，腫瘤細胞於體內快速的異常增生，當分化並成熟至一定程度時，會開始進行上皮間質轉化過程(epithelial-to-mesenchymal transition, EMT)。其在惡性腫瘤的侵犯及轉移中扮演著相當重要的機制，他會促使腫瘤細胞進入身體循環系統，形成循環腫瘤細胞(circulating tumor cells, CTCs)，使腫瘤藉著血液或淋巴循環，散佈至身體其他區域。而其中上皮間質轉化細胞(EMT-derived cells)和循環腫瘤幹細胞(circulating cancer stem cells, CCSCs)被認為是現今癌症治療中最棘手的角色之一，不只促使腫瘤生長、侵犯、轉移，且因具有自我更新及分化的能力，易造成復發及抗藥性，導致治療失敗。
已知在腫瘤微環境中，不論是透過腫瘤壞死因子(TNF-α)、轉化生長因子(TGF-β)、肝細胞生長因子(HGF)、纖維母細胞生長因子(bFGF)、上皮生長因子(EGF)、或是HIF-1α、HMGA2…等機制去誘導上皮間質轉化的發生，都會進一步誘導Twist轉錄因子的表達上升。因此，本研究選擇EMT的關鍵調節分子Twist轉錄因子，當作分子標誌物(molecular marker)去尋找腫瘤中可能之上皮間質轉化細胞，搭配特異性結合之tLyP-1 CendR motif胜肽，並使用適量薑黃素(curcumin)誘導，進一步優化出偵測上皮間質轉化細胞及血液中循環腫瘤幹細胞的方法。
根據初步的研究結果發現，利用我們建構的Twist啟動子連結雙融合報導基因系統搭配特異性結合之tLyP-1 CendR motif胜肽，在頭頸部癌症SAS細胞株中具有偵測上皮間質轉化細胞的特性。且經過適量薑黃素誘導之後，在小鼠活體內及血液中也同樣可以利用分子醫學影像偵測出腫瘤中可能的上皮間質轉化細胞，相信對未來惡性腫瘤的追蹤、標靶治療、轉移以及復發等研究皆能有所助益。

Malignant tumors are a highly heterogeneous tissue with rapid and abnormal proliferation. When they differentiate and mature to a certain extent, epithelial-to-mesenchymal transition (EMT) begins. EMT plays a very important mechanism in the invasion and metastasis of malignant tumors, which promotes the tumor cells to enter circulation systems and form circulating tumor cells (CTCs). EMT-derived cells and circulating cancer stem cells (CCSCs) are considered to be one of the most difficult roles in cancer treatment today, not only promote tumor growth, invasion and metastasis, but also induce self-renewal and differentiation. Therefore, EMT-derived cells and CCSCs are believed to be resistant to currently available therapies and may be responsible for relapse of cancer in patients.
According to the previous studies, EMT can be induced through the tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), fibroblast growth factor (bFGF), epithelial growth factor (EGF), HIF-1α, or HMGA2, and will further upregulate Twist transcription factor expression. Consequently, we selected Twist transcription factor, a key regulator of EMT, as a molecular marker to search for possible EMT-derived cells in tumors. Meanwhile, we tried to induce and capture CTCs by using an appropriate dose of curcumin and specific binding peptides, tLyP-1, and intended to optimize the method for detecting EMT-derived cells and CCSCs in the blood.
Our preliminary results showed that using this Twist reporter gene system combined with the specific binding peptide is able to detect EMT-derived cells in head and neck cancer-SAS cell line. After induction with an appropriate dose of curcumin, EMT-derived cells of the tumors can be detected both in vivo and in the blood of mice. The novel technology we developed is not only able to track metastatic and relapsed cancer cells, but also beneficial for developing of novel targeted therapies of cancers.

目錄
致謝 i
目錄 ii
中文摘要 iv
英文摘要 i
一、 前言 1
1. 腫瘤中的上皮間質轉化 (epithelial-to-mesenchymal transition) 細胞 1
2. 循環腫瘤細胞 (circulating tumor cell, CTC) 2
3. 腫瘤中的上皮間質轉化與其相關分子機制 2
4. 利用分子影像標記可能之上皮間質轉化細胞 4
5. 利用薑黃素誘發上皮間質轉化細胞及循環腫瘤細胞 5
6. 神經纖毛蛋白質及其腫瘤標靶胜肽 6
二、研究目的 8
三、材料與方法 10
1. 質體建構 (Plasmid establishment) 10
2. 細胞培養 (Cell culture) 11
3. 活體外細胞載體轉染 (In vitro transfection) 11
4. 建立穩定轉染之細胞株 (Stable clone establishment) 12
5. 活體外細胞螢光試驗 (In vitro functional test of fluorescence assay) 12
6. 活體外細胞攝取放射性示蹤劑[3H]-FEAU試驗 ([3H]-FEAU uptake assay) 13
7. 蛋白質濃度測定 (Protein concentration determination) 13
8. 生長曲線 (Growth curve) 14
9. 胜肽特異性結合測試 (Peptide specific binding assay) 14
10. 動物模式之建立 (Animal model) 15
11. 活體小動物正子斷層影像 15
12. 活體小動物生物螢光影像 15
13. 薑黃素誘導循環腫瘤細胞富集 (Curcumin induce CTCs enrichment) 16
14. 冷凍切片 (Cryosection) 16
15. 免疫螢光染色 (Immunofluorescence) 16
16. 統計分析 17
四、研究結果 18
1. 建構Twist啟動子之雙融合報導基因質體 18
2. Twist啟動子雙報導基因功能測試 18
2-1. Twist啟動子受到HIF1α-ODD誘導而啟動報導基因表現 18
2-2. Twist啟動子受到v-Src誘導而啟動報導基因表現 19
3. 建立受HIF-1α-ODD 誘導的穩定轉染細胞株及之功能性測試 20
3-1. SAS-pTwist-E2-Crimson-P2A::ttksr39之建立 20
3-2. SAS-pCMV-E2-Crimson-P2A::ttksr39之建立 21
4. SAS及其穩定轉染細胞株之生長曲線 22
5. 活體外細胞胜肽結合測試 (In vitro cancer stem cell peptide binding assay) 22
6. 活體影像 23
6-1. 活體螢光影像 (PhotonIMAGER ™ Optima) 23
6-2. 多功能動物分子影像 (FLEX Triumph TM) 24
6-3. 腫瘤組織之螢光影像分析 (PhotonIMAGER ™ Optima) 25
6-4. 血液之螢光影像分析 (PhotonIMAGER ™ Optima) 25
7. 雷射共軛焦顯微鏡 (Zeiss LSM 880) 25
7-1. 腫瘤組織之切片及免疫螢光染色觀察 26
8. 流式細胞儀 (Beckman Coulter CytoFLEX) 26
五、結論與討論 28
六、參考文獻 31
七、附圖 35

六、參考文獻
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