臺灣博碩士論文加值系統

在先前的研究中發現癌細胞在2D和3D不同的環境中，會展現出不同的細胞特性。而且微環境對於癌細胞的幹細胞特性具有重要的影響作用。在微環境中，腫瘤相關纖維母細胞 (CAF) 比一般纖維母細胞更能促進腫瘤的發展。因此，本研究目的為探討癌細胞在立體環境中，纖維母細胞對於癌症幹細胞特性的影響。

實驗使用的細胞為鼻咽癌細胞珠TW01、TW06和Hone-1，以及癌症幹細胞TW01 CSC進行實驗。纖維母細胞則是來自於肺癌病患的腫瘤組織以及正常組織，給予細胞介白素6 (interleukin 6，IL-6) 或乳糖以誘發成CAF，藉由觀察CAF markers如: alpha-smooth muscle actin (α-SMA) 和vimentin以進行確認。將細胞培養於第一型膠原蛋白所建構的立體環境中，利用西方點墨法分析癌細胞的幹細胞相關基因Oct-4和Nanog的表現。

由實驗結果發現，在3D模型中癌細胞會形成聚球 (sphere) 的結構顯示相較於2D環境，細胞能展現更多的幹細胞特性。在立體環境中，癌細胞會有較高的Nanog表現，然而CSC組別中，Nanog表現卻是下降的。而在纖維母細胞的實驗中，證實乳糖有如IL-6具有促使一般纖維母細胞轉變為CAF的作用。接著將癌細胞與CAF在3D 環境中共同培養，發現CAF能促使癌細胞Nanog有比較高的表現，尤其是與乳糖誘發的CAF共同培養的組別。而且CSC組別中，與乳糖誘發的CAF培養後，Oct-4和Nanog都有顯著的增加情形。顯示微環境以及其中所存在的CAF對癌細胞的幹細胞特性能產生影響。

Previous studies revealed that stemness properties of cancer cells are different whether they are in 2D or 3D culture environment. Microenvironment of cancer cells has important roles with the stemness of cancer. In addition, cancer-associated fibroblasts (CAFs) existing in the tumor microenvironment have better ability to promote tumor development than normal fibroblast. Therefore, we compared the influences on NPC stemness properties with different fibroblast in 3D culture system.
NPC cell lines, TW01, TW06, Hone-1 and TW01 CSC, were used in this study. Fibroblasts were cultured from patients’ normal tissue or tumor tissue. Interleukin 6 (IL-6) and lactate were used to induce normal fibroblast to CAF and alpha-smooth muscle actin (α-SMA) and vimentin as the CAF markers were analyzed. Collagen type I was used as matrix to construct 3D culture environment. Expression of stemness markers, Oct-4 and Nanog, were assayed by Western blot.
In 3D system, NPC cells could form more sphere structure than in 2D culture, that indicated the stemness of cells was increased in 3D system. Nanog expressed more in 3D mono-culture than 2D system by western blot. However, Nanog was decreased in cancer stem cell (CSC) of NPC. In the other side, CAF markers were increased after treated with IL-6 or lactate in normal fibroblasts, as the previous research. When NPC cells were co-cultured with CAF, the protein level of Nanog was increased. In addition, Oct-4 and Nanog both enhanced, when CSC co-cultured with fibroblast treated with lactate. Furthermore, the concentration of lactate which released to medium rose if NPC cells/CSC co-cultured with CAF. In this study, we found that 3D culture environment with fibroblasts might change stemness properties of cancer cells.

誌謝 i
中文摘要 ii
Abstract iii
目錄 (Contents) iv
緒論 (Introduction) - 1 -
鼻咽癌 (Nasopharyngeal carcinoma) - 1 -
癌症幹細胞 (Cancer stem cell，CSC) - 1 -
鼻咽癌幹細胞 (Cancer stem cell of Nasopharyngeal carcinoma) - 2 -
腫瘤微環境 (Tumor microenvironment，TME) - 3 -
腫瘤相關纖維母細胞 (Cancer-Associated Fibroblast，CAF) - 3 -
癌細胞的代謝路徑 (The metabolism of cancer cell) - 4 -
(a) Warburg effect - 4 -
(b) Reverse Warburg Effect - 5 -
乳糖在癌症中扮演的角色 (The role of lactate in cancer) - 6 -
3D培養模型 (Three-dimensional culture model) - 6 -
研究目標 (Specific Aims) - 8 -
材料與方法 (Material and Methods) - 9 -
使用的細胞株 - 9 -
細胞培養 - 9 -
誘發纖維母細胞轉變成腫瘤相關纖維母細胞 - 9 -
3D 細胞培養模型 - 10 -
蛋白質萃取 - 10 -
西方點墨法 (Western blot) - 10 -
免疫螢光染色法 (Immunofluorescence staining) - 11 -
共軛焦顯微成像技術 (Confocal microscopy) - 11 -
乳糖濃度分析 (Lactate releasing assay) - 12 -
統計 (Statistical analysis) - 12 -
表格一 (Table 1) - 12 -
表格二 (Table 2) - 13 -
結果 (Result) - 14 -
分析來自不同組織部位的肺癌病人檢體 - 14 -
乳糖以及IL-6能誘發纖維母細胞表現CAF marker - 14 -
給予乳糖後持續培養的纖維母細胞能維持CAF markers的表現 - 14 -
培養於3D culture的鼻咽癌細胞及癌症幹細胞釋放的乳糖濃度較2D culture高 - 15 -
在3D culture中，與腫瘤相關纖維母細胞共同培養的癌細胞，其培養液有較高濃度的乳糖 - 15 -
TW01細胞株在2D和3D culture下會展現不同的細胞型態 - 15 -
透過將α-tubulin染色以觀察TW01細胞株的型態 - 16 -
分析鼻咽癌細胞珠的幹細胞相關基因表現量 - 16 -
鼻咽癌幹細胞之幹細胞相關基因表現量 - 16 -
在3D Culture中，與纖維母細胞共同培養的TW01細胞株其幹細胞相關基因表現量 - 17 -
在3D culture中，將癌細胞與施予過IL-6或乳糖的纖維母細胞培養過後，Nanog表現量有顯著性的增加 - 17 -
在3D culture中，將癌症幹癌細胞與給予乳糖的纖維母細胞培養過後，Oct-4及Nanog表現量有顯著性的增加 - 17 -
討論 (Discussion) - 18 -
參考文獻 (Reference) - 20 -
圖 (Figure) - 25 -

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