根據中華民國100年行政院衛生署十大癌症死因統計，肝癌於不分男女之排名中佔癌症主要死因第二位，更高居全世界排名的第四位，所以在臺灣如何維護肝臟健康是值得重視的議題。近年研究發現，臺灣特有之牛樟芝具有護肝、抗發炎、抗B型肝炎病毒、抗癌等生理活性，其主要活性成分為多醣體、苯類、三萜類、固醇類等，又因三萜類通常具有良好的抗癌效果而備受矚目。因此，本研究欲探討椴木栽培之牛樟芝子實體中含量第二多的三萜類──齒孔酸對人類肝癌Hep 3B細胞株之抗癌活性及其分子機制。結果指出，齒孔酸能有效抑制Hep 3B細胞存活率，其處理24小時之半抑制濃度為18.4 μM，相當於8.7 μg/mL。除此之外，齒孔酸會促使Hep 3B細胞中的LC3-Ⅰ轉變為LC3-Ⅱ，並產生大量的自噬體和自噬溶酶體，但不會明顯地增加細胞的亞二倍體比例或導致細胞破損，故齒孔酸誘導Hep 3B細胞死亡之主要模式為自體吞噬，而不是細胞凋亡或細胞壞死。深入探討其分子機制後發現，齒孔酸首先可促進Hep 3B細胞中的ROS生成和降低ATP水平而導致內質網壓力產生，接著促使細胞質中的鈣離子濃度和BiP表現量上升、DAPK的磷酸化下降及Beclin-1、JNK和Bcl-2的磷酸化上升，最終導致自體吞噬。綜合以上結果顯示，齒孔酸的抗肝癌功效相當良好，且機制較為獨特，加上其在椴木栽培之牛樟芝子實體中含量甚高，因此具有量產並輔助抗癌治療的潛力。

　　Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics, and ranks the fourth in cancer related mortality in the world. Hence to maintain a healthy liver is a big issue in Taiwan. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, can manipulate biological activities, including hepatoprotection, anti-inflammation, anti-HBV activity, anticancer activity, etc. The active constituents include polysaccharides, benzenoids, triterpenoids, steroids, etc., and among them triterpenoids are the most prominent because of their potent anticancer effects. In this study, the anti-liver cancer activity and molecular mechanisms of eburicoic acid, the second most abundant triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea was investigated using the human hepatoma Hep 3B cells. The results show that eburicoic acid effectively reduced Hep 3B cell viability within 24 hours, and the IC50 was 18.4 μM, which was equivalent to 8.7 μg/mL. Besides, eburicoic acid induced conversion of LC3-Ⅰto LC3-Ⅱ and a large number of autophagosomes/autophagolysosomes formation, but increasing of hypodiploid proportion or cell lysis obviously in Hep 3B cells. So the principal mode of Hep 3B cell death induced by eburicoic acid was autophagy, rather than apoptosis or necrosis. In depth investigation for the molecular mechanisms, revealed that eburicoic acid firstly promoted ROS generation and ATP depletion, leading to ER stress, followed by elevated cytosolic calcium ion concentration and BiP expression, downregulated phosphorylation of DAPK, upregulated phosphorylation of Beclin-1, JNK, and Bcl-2, and finally induced autophagy in Hep 3B cells. These results indicate that eburicoic acid has significant anti-liver cancer effects and more distinctive mechanisms. Coupled with these findings and the high content of eburicoic acid in the fruiting bodies of basswood cultivated Antrodia cinnamomea, eburicoic acid has the potential for mass production and to assist cancer therapy.

致謝...I
摘要...II
Abstract...III
縮寫對照表...V
目錄...VIII
表次...XII
圖次...XIII
第一章　文獻回顧...1
　第一節　肝癌...1
　　一、肝臟生理功能...1
　　二、肝癌流行病學...4
　　三、肝癌臨床病理...6
　第二節　細胞死亡模式...8
　　一、細胞凋亡形態特徵...8
　　二、自體吞噬形態特徵...10
　　三、細胞壞死形態特徵...12
　　四、細胞凋亡誘導路徑...12
　　五、自體吞噬誘導路徑...14
　第三節　內質網壓力...17
　　一、內質網生理功能...17
　　二、內質網壓力反應...18
　　三、內質網壓力連結自體吞噬之路徑...19
　第四節　牛樟芝...21
　　一、牛樟芝形態與命名...21
　　二、牛樟芝人工栽培方法...23
　　三、牛樟芝生理功效...24
　　四、牛樟芝活性成分...29
第二章　動機與目的...37
第三章　研究架構...38
第四章　材料...39
　第一節、樣品與細胞株...39
　　一、樣品...39
　　二、細胞株...39
　第二節、藥品與儀器...40
　　一、藥品...40
　　二、儀器...42
第五章　方法...45
　第一節、抗增殖活性評估...45
　　一、細胞之培養與保存...45
　　二、細胞存活率分析...46
　第二節、細胞死亡模式分析...47
　　一、細胞形態觀察...47
　　二、細胞大小及顆粒性分析...47
　　三、亞二倍體比例分析...48
　　四、細胞破損程度分析...49
　　五、LC3分布觀察...50
　　六、自噬體形成量分析...51
　第三節、內質網壓力調節之自體吞噬機制探討...52
　　一、活性氧物質生成分析...52
　　二、三磷酸腺苷水平分析...53
　　三、鈣離子移位分析...53
　　四、蛋白質表現分析...55
　第四節、資料統計...57
第六章　結果與討論...58
　第一節、抗增殖活性評估...58
　　一、Eburicoic acid對Hep 3B細胞存活率之影響...58
　　二、本節討論...59
　第二節、細胞死亡模式分析...60
　　一、Eburicoic acid對Hep 3B細胞形態之影響...60
　　二、Eburicoic acid對Hep 3B細胞亞二倍體比例之影響...61
　　三、Eburicoic acid對Hep 3B細胞破損程度之影響...61
　　四、Eburicoic acid對Hep 3B細胞自噬體形成之影響...62
　　五、本節討論...62
　第三節、內質網壓力調節之自體吞噬機制探討...64
　　一、Eburicoic acid對Hep 3B細胞活性氧物質及三磷酸腺苷之影響...64
　　二、Eburicoic acid對Hep 3B細胞鈣離子訊號之影響...65
　　三、Eburicoic acid對Hep 3B細胞不完全摺疊蛋白反應之影響...65
　　四、本節討論...66
　第四節、綜合討論...68
　　一、Eburicoic acid與dehydroeburicoic acid之抗癌機制比較...68
　　二、Eburicoic acid與牛樟芝子實體萃取物之抗癌機制比較...69
　第五節、未來研究...70
　　一、Eburicoic acid之後續抗癌研究...70
　　二、牛樟芝子實體中麥角甾烷型三萜類之後續研究...70
第七章　結論...72
第八章　實驗圖表...74
第九章　參考文獻...86
第十章　附錄...94

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