臺灣博碩士論文加值系統

肝癌在全球為排名第三的惡性腫瘤，超過75% 的肝癌病患出現在亞太地區。在台灣每年約有3700位病人因肝癌而死亡。目前有許多的化學物藥物已經使用在 肝癌的治療上。牛樟芝為醫藥用真菌且在台灣被當作保健食品，先前文獻指出牛樟芝萃取物可以抑制腫瘤細胞生長、維護肝臟功能、抗發炎及抗氧化等功能。然而牛樟芝對於肝癌患者在接受藥物治療的安全評估不清楚。因此牛樟芝萃取物對藥物誘導肝癌細胞死亡的影響需要去確認。
本研究想要去探討牛樟芝萃取物對於藥物處理肝癌細胞時所扮演的角色和機制影響。首先我們以酒精來萃取牛樟芝培養基 (EAC)，初步結果顯示當給予高濃度的藥物 (5-25 M) 或牛樟芝萃取物 (200-250 g/ml) 處理48小時後可以明顯抑制Huh-7和HepG2肝癌細胞生長，而當以藥物合併EAC處理時發現EAC可以明顯增強Huh-7和HepG2細胞對低濃度藥物的敏感度。而在細胞的外觀上，藥物合併EAC處理時可以使Huh-7細胞從平貼變成圓形。這初步結果顯示EAC對於藥物在處理肝癌細胞時扮演重要角色。因此在本論文中我們進一步分析藥物合併EAC對Huh-7細胞週期的影響，G2 / M期細胞的百分比顯著增加；再更進一步分析Hun-7細胞週期相關蛋白質，經合併處理有抑制現象。在動物模式上，藥物合併EAC明顯抑制腫瘤生長。這樣的結果可以提供一個重要的牛樟芝保健食品安全資訊給在以藥物治療肝癌的病人。

Hepatocellular carcinoma (HCC) is the third most common malignancy worldwide. More than 75% of HCC cases occur in the Asia-Pacific region. In Taiwan, there are about 3,700 patients a year die from liver cancer. There are many chemical drugs have been used in liver cancer therapy. Antrodia cinnamomea is a medicinal fungus that has been served as a functional food in Taiwan for decades. Previous studies have shown that the secondary metabolites form Antrodia cinnamomea have various activities such as antitumor, protection of liver function, anti-inflammation and antioxidant. However, the safety and the possible interaction of Antrodia cinnamomea extracts with drugs in liver cancer patients have been not studied yet.
In the current project, we want to identify the possible role and mechanisms of Antrodia cinnamomea extracts in modulating the activity of drugs in hepatoma cells both in vitro and in vivo. First, Antrodia cinnamomea extracts (EAC) were isolated from culture medium using ethanol. Our preliminary results indicated that the exposure to high concentrations of drugs (5-25 M) or Antrodia cinnamomea extracts (200-250 g/ml) for 48 h caused significant death in Huh-7 and HepG2 cells. In addition, EAC could sensitive tumor cells to low dose of drugs in Huh-7 and HepG2 cells.Considering the cell morphology, the combination of drugs with EAC caused progressive changes in Huh-7 cells from flat to round. Therefore, our preliminary results indicated that EAC could have a positive role in the application of drugs for the treated hepatocellular carcinoma. In this paper, we will further analyze the effect of combination on cell cycle for Huh-7 cells. The percentage of G2/M phase cells notably increased. Then further analysis of cell cycle related proteins on Hun-7 cells, the combination-treated has inhibition. In animal models, the combination significantly inhibition of tumor growth. These results could provide important insight in chemotherapy and safety of Antrodia cinnamomea.

中文摘要 I
Abstract III
目錄 IV
圖目錄 VII
附錄 XI
英文縮寫對照表 XII
第一章 緒論 1
1.1肝癌 1
1.2 Sorafenib (Nexavar，蕾莎瓦) 5
1.3 牛樟芝萃取物 ( Antrodia cinnamomea extracts , EAC ) 8
1.4 細胞週期 (Cell cycle) 13
1.5 細胞凋亡 (Apoptosis) 21
1.6 絲裂原活化蛋白激酶家族 (MAPKs) 途徑與細胞凋亡 28
1.7 研究動機 31
第二章 材料與方法 33
2.1 材料 33
2.1.1 藥品 33
2.1.2 器材 35
2.2 牛樟芝菌絲體培養與萃取 35
2.3 細胞培養 35
2.3.1 細胞培養條件 35
2.3.2 細胞繼代培養 36
2.3.3 計數細胞 36
2.3.4 冷凍細胞 37
2.3.5 解凍細胞 37
2.4 細胞存活率 (MTT assay) 38
2.5 細胞外型觀察 39
2.5.1 單一藥物處理 39
2.5.2 兩種藥物合併處理 39
2.6 螢光染色 39
2.7 細胞週期的分析 (Flow cytometry，流式細胞儀) 40
2.8 Caspase-3 分析 (Flow cytometry，流式細胞儀) 41
2.9 Apoptosis 分析 (Flow cytometry，流式細胞儀) 42
2.10 傷口癒合實驗 (Wound Healing assay) 43
2.11 西方墨點法 43
2.11.1 細胞內蛋白質萃取 43
2.11.2 蛋白質定量 44
2.11.3 膠體配製 45
2.11.4 蛋白質電泳 47
2.11.5 蛋白質轉漬 47
2.11.6 免疫墨點法 48
2.12 腫瘤抑制動物模式 49
第四章 討論 59
第五章 結論 64
參考文獻 65
圖 76
附錄 98

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