臺灣博碩士論文加值系統

摘要
在衛生福利部最新釋出的 2018 癌症發生率，大腸癌 (Colorectalcancer) 發 生率位居第一，當體內存在過多活性氧物質時，會產生氧化壓力和發炎作用，並 活化 Cyclooxygenase (COX-2) 和 Nuclear factor-kappa B (NF-κB)，導致促炎激 素分泌量異常增加，進而促進腫瘤的產生。已有研究指出褐藻中萃取出的硫酸多 醣具有抗氧化、抗發炎、抗癌等功效，已經在不同類型的癌症中體內和體外證明 了硫酸多醣的抗癌特性。本研究使用半葉馬尾藻 Sargassumhemiphyllum 水萃物 (SHw) 探討其對大腸癌體內發炎環境之改善效果，首先測量 SHw 的主成分以 確定硫酸根和褐藻醣膠含量分別為 61.9 ± 0.16 及 439.6mg/g。在細胞實驗中半 葉馬尾藻水萃物對小鼠大腸癌細胞 CT26.WT 有毒殺效果;在 LPS 誘導的巨噬 細胞 RAW264.7 中 SHw 良好的抑制自由基 NO 的產生;接著再將 CT26.WT 細胞皮下注射到 BALB/c 及使用 Azoxymethane (AOM)/Dextran sodium sulfate (DSS) 誘導大腸癌作為動物模式。半葉馬尾藻水萃物有效改善腫瘤的數量及體積 及降低血液中的 Reactiveoxygenspecies(ROS) 和發炎激素含量，並藉由抑制自 由基和 COX-2 進而減少 Hypoxia-inducible factor 1-alpha (HIF-1α) 及 Vascular endothelial growth factor (VEGF) 來改善血管新生，最後在組織切片中觀察到 SHw 對於腸道隱窩有改善效果，這些結果證明半葉馬尾藻水萃物具有抗癌活性。

Abstract
Colorectal cancer was the highest occurrence in the 2016 cancer incidence rate which was reported by the Ministry of Health and Welfare, Taiwan. Recent studies indicated that an excessive reactive oxygen species (ROS) transforms oxidative stress and inflammation. oxidative stress activates Oxidative stress activates cyclooxygenase (COX-2) and nuclear factor-kappa B (NF-κB), resulting in the abnormal pro- inflammatory cytokines secretion, thereby promoting tumor growth. It has been reported that sulfated polysaccharides of fucoidan from brown algae has anti-oxidative, anti-inflammatory, anti-cancer effects and proved in vivo and in vitro in different types of cancer. In this study, we investigated into the protection of Sargassum hemiphyllum water extract (SHw) in the colorectal cancer. First, the main components of SHw measured to the sulfate and fucoidan contents of 61.9 ± 0.16 and 439.6 mg/g, respectively. In the cell-line experiment, the SHw was toxic to CT26.WT; SHw inhibited the production of NO in LPS-induced macrophage RAW 264.7. We also evaluated the effects of SHw on CT26.WT-bearing mouse and Azoxymethane (AOM)/Dextran sodium sulfate (DSS) induced colorectal cancer as an animal model. The SHw effectively improved the number and volume of tumors, as well as reduced ROS and pro-inflammatory cytokines. Therefore, downstream factors, i.e. hypoxia- inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF) were down regulated by means of decreased ROS and COX-2, thus improving angiogenesis. Finally, SHw was observed to improve the intestinal crypt in tissue sections. In summary, our results revealed that SHw is able to enhance anticancer activity.

目錄
壹、文獻回顧 2
一、癌症的起源 2
二、大腸癌 2
1. 大腸癌病理學 2
2. 現今結腸癌治療方式 3
三、炎症性免疫反應 4
四、囓齒動物大腸癌 5
1. 同種移殖 (Allogeneic Model) 5
2. 異種移植 (Xenograft model) 6
五、海藻-褐藻 (Phaeophyceae) 8
1. 海藻 (Seaweed) 8
2. 褐藻 (Phaeophyta) 9
3. 半葉馬尾藻 (Sargassum hemiphyllum) 9
4. 褐藻醣膠 (Fucoidan) 9
貳、實驗設計流程 11
一、褐藻萃取 11
二、細胞實驗 11
三、動物實驗 12
參、實驗材料與方法 14
一、實驗材料 14
1. 樣品原料 14
2. 誘導腸癌之化學品 14
3. 標準品 14
4. 抗氧化試驗 14
5. 細胞株 14
6. 細胞培養之培養基 14
7. 細胞存活率試劑 15
8. 一氧化氮生成試驗 15
9. 實驗動物 15
10. 組織切片 15
11. 蛋白質萃取與西方墨點法相關試劑 16
12. 細胞激素分析 16
13. Reactive oxygen species (ROS) 分析 16
二、儀器設備 17
三、實驗方法 17
1. 萃取物製備 17
2. 萃取物之結構與組成鑑定及物化性分析 17
3. 細胞實驗 (In vitro) 20
4. 細胞毒性測試 (Cytotoxicity test) 20
5. 細胞內一氧化氮 (Nitrate oxide, NO) 含量測定 21
6. 小鼠大腸癌動物模式之建立 21
8. 血液內粒線體 ROS 含量測定 22
8. ELISA 22
9. 蛋白質定量分析 22
10. 病理組織切片 24
11. 統計分析 25
肆、結果 26
一、半葉馬尾藻萃取與特性分析 26
1. 半葉馬尾藻萃取物之產率及成分分析 26
2. 半葉馬尾藻萃取物之總醣、硫酸根含量及抗氧化能力測定 26
二、半葉馬尾藻萃取物對小鼠大腸癌細胞株 (CT26. WT) 及小鼠巨噬細胞株RAW264.7 抗氧化之影響 26
1. 半葉馬尾藻萃取物對於 CT26. WT 及 BHK-21 之細胞毒性試驗 26
2. 半葉馬尾藻萃取物對於小鼠巨噬細胞株 RAW264.7 之細胞毒性試驗及利用 LPS 誘導一氧化氮之影響 27
三、半葉馬尾藻萃取物對大腸癌動物模式之探討 27
1. 皮下注射大腸癌細胞之動物模式 27
2. 化學誘導大腸癌細胞之動物模式 29
伍、討論 31
一、 半葉馬尾藻萃取物成分與生理活性 31
二、半葉馬尾藻萃取物體外實驗 32
二、 探討半葉馬尾藻萃取物對於大腸癌動物模式之影響 33
陸、結論 36
柒、參考文獻 37
捌、圖 45
玖、表 62

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