臺灣博碩士論文加值系統

肝癌為世界第二大癌症；癌症死亡率第三的癌症，許多藥物被用來治療肝癌如doxorubicin或sorafenib等藥物。本研究中我們的目的主要為探討牛樟芝萃取液(EAC)對於Sorafenib誘導肝癌細胞死亡機制的影響。當合併EAC與sorafenib處理時顯著抑制Huh-7和HepG2的生長，而在Western blot分析出多種訊號顯示合併藥物處理可以調節MAPK和NF-B的路徑達到抗腫瘤的功效，也會誘導細胞週期蛋白表達的改變。以傷口癒合試驗探討合併藥物治療對Huh-7細胞遷移機制的影響；以real time-PCR檢測合併治療對Huh-7細胞的轉移相關蛋白的表現。並且以動物試驗探討合併藥物治療對於肝腫瘤生長的影響。發現合併治療有抑制MAPK家族及細胞週期相關蛋白的表達、抑制增殖能力和降低遷移相關蛋白的表現量。結果顯示EAC能增強Sorafenib誘導細胞死亡能力、增加肝癌細胞對sorafenib的敏感度及抑制肝癌細胞的遷移能力，依此結果運用在肝癌治療中建議EAC可以降低Sorafenib藥量，對於因過量使用Sorafenub患者而產生的副作用的患者，將有相當大的助益，對於未來將進一步探討Sorafenib在EAC的輔助下其更多的機制。

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer
worldwide and the third-leading cause of cancer death, and varied drugs were
used to treatment in HCC such as doxorubicin or sorafenib. In the present study, we want to investigate the effects Antrodia Cinnamomea extracts (EAC) on the anticancer activity of sorafenib treatment in HCC cells. First of all, combination treatment resulted in significant growth inhibition in HepG2 and Huh-7 cells.
Western blot analysis of various signaling markers revealed the ability of
sorafenib with EAC to target pathways mediated by mitogen-activated protein
(MAP) kinase and NF-kB, of which the concerted action underlined its
antitumor efficacy. The combination treatment also induced alternation of cyclin proteins expression. Besides, the Wound Healing assay were perform to study the influence of combined drugs treatment on Huh-7 cells migration mechanism.
After that, the MMP-2 and MMP-9 Protein expression of combined drugs treatment on Huh-7 was detected by Quantitative PCR. Finally, in animal model assay were performed to study the influence of combined drugs treatment on HCC tumor growth and protein expression. We found that combination IV significantly inhibition of MAPK family and cell cycle protein expression, inhibition of cell proliferation and reduce protein expression of migration. Our results indicate that EAC can enhance sorafenib for the treatment of HCC cells and enhance sensitivity of sorafenib and inhibition of migration in HCC cells.These results suggest that the EAC is very helpful to reduce the amount of sorafenib, and decreased the side effects of sorafenib on patients in further. We will be further investigating sorafenib in the auxiliary EAC its additional mechanisms.

中文摘要 I
Abstract II
誌謝 IV
目錄 VI
圖目錄 VII
附錄目錄 XIII
英文縮寫對照表 XIV
第一章 緒論 17
1.1肝癌 ( Liver cancer ) 17
1.2 Sorafenib (蕾莎瓦) 20
1.3 牛樟芝萃取物 ( Anrodia cinnamomea extracts , EAC ) 21
1.4細胞凋亡 ( Apoptosis ) 24
1.5 絲裂原活化蛋白激酶家族 ( MAPKs ) 細胞凋亡途徑 27
1.6 基質金屬蛋白酶家族 ( Matrix metalloproteinases , MMPs ) 29
1.7 研究動機 30
第二章 材料與方法 31
2.1材料 31
2.1.1藥品 31
2.1.2器材 33
2.2牛樟芝菌絲體培養與萃取 33
2.3細胞培養 34
2.3.1 細胞培養條件 34
2.3.2細胞繼代培養 34
2.3.3 計數細胞 35
2.3.4冷凍細胞 35
2.3.5解凍細胞 36
2.4 細胞外型觀察 36
2.4.1 單一藥物處理 36
2.4.2 兩種藥物合併處理 36
2.5 細胞存活率 (MTT assay) 37
2.6 細胞週期的分析 ( Flow cytometery，流式細胞儀 ) 38
2.7 Caspase-3 分析 ( Flow cytometery，流式細胞儀 ) 39
2.8 Apoptosis 分析 ( Flow cytometery，流式細胞儀 ) 40
2.9 西方墨點法 41
2.9.1小鼠肝臟腫瘤組織內蛋白質萃取 41
2.9.2 細胞內蛋白質萃取 41
2.9.3蛋白質定量 42
2.9.4電泳膠體配置 43
2.9.5 蛋白質電泳 43
2.9.6 蛋白質轉漬 44
2.9.7免疫墨點法 44
2.10 螢光染色 45
2.11 傷口癒合實驗 (Wound healing assay) 46
2.12 抽取細胞培養total RNA (Isolate total RNA) 46
2.13 反轉錄聚合酶連鎖反應 (RT-PCR ) 47
2.14即時聚合酶連鎖反應 ( Real-time PCR ; Q-PCR ) 47
第三章 結果 49
3.1牛樟芝皿培式培養及萃取 49
3.2 Sorafenib、EAC單獨處理Huh-7、G2的細胞存活率測試 49
3.3 Sorafenib配合EAC處理Huh-7、G2的細胞存活率測試 50
3.4 Sorafenib、EAC對Huh-7細胞型態的變化 51
3.5 Sorafenib配合EAC對Huh-7細胞型態的變化 51
3.6 Sorafenib配合EAC對於Huh-7細胞內蛋白質的表現量 52
3.7 Sorafenib配合EAC對於Huh-7細胞凋亡的影響 53
3.8 Sorafenib配合EAC對於Huh-7細胞爬行能力影響 54
3.9 Sorafenib配合EAC對於Huh-7細胞內MMP-2/9影響 54
3.10 Sorafenib配合EAC對於動物模式的Huh-7腫瘤抑制能力 55
3.11 Sorafenib搭配EAC對於小鼠腫瘤組織內蛋白質的表現量 56
3.12 Sorafenib 搭配 EAC 是否與搭配子實體牛樟芝有相同的功效。 56
第四章 討論 58
第五章 結論 63
參考文獻 64
附圖 71
附錄 104

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