臺灣博碩士論文加值系統

惡性腫瘤位居國人十大死因之首，肝癌更是國人十大癌症死因中的第二名。隨著肝癌之發展惡化，轉移是導致癌症病患死亡之主因。因此就台灣而言，如何預防肝癌並且抑制肝癌的轉移是一個值得重視的議題。有文獻指出牛樟芝子實體乙酸乙酯萃取物（EAC）能夠抑制人類肝癌細胞的侵犯能力，本實驗所使用的antcin K為牛樟芝子實體的主要活性成分，麥角甾烷型三&;#33820;類純化物中含量最高者，因此本研究之假說推測antcin K具有抑制人類肝癌細胞轉移的功效。本研究目的為評估antcin K抑制人類肝癌細胞轉移之效果及其機制探討。結果顯示在antcin K的處理下，可顯著降低Hep 3B細胞黏附於細胞外基質的作用以及移行和侵犯的能力，並且可以降低Hep 3B細胞內基質金屬蛋白&;#37238;─2（MMP-2)和基質金屬蛋白&;#37238;─9（MMP-9)的蛋白質表現量以及其分泌活性，並促使Hep 3B細胞中上皮細胞指標蛋白E-cadherin表現量上升，而間質細胞指標蛋白vimentin表現量下降。更深入探討其相關分子機制發現，antcin K可以降低Hep 3B細胞內Integrin β1、β3、α5及αv的蛋白質表現量，並且抑制FAK、Src、PI3K、AKT、MEK、ERK及JNK蛋白質的磷酸化。綜合以上結果推測antcin K可能是藉由降低細胞中Integrin β1、β3、α5及αv的蛋白質表現量及FAK的活化，並且抑制下游PI3K/AKT、MEK/ERK及JNK訊息傳遞途徑的活化，進而降低細胞的MMP-2和MMP-9蛋白質表現量以及其分泌活性，同時影響上皮─間質轉化（epithelial-mesenchymal transitions, EMT)，最後達到抑制人類肝癌細胞轉移的效果。本研究結果指出椴木栽培牛樟芝子實體中麥角甾烷三&;#33820;類活性成分antcin K具有降低肝癌轉移風險的潛力，期待其將來能作為抑制癌細胞轉移和輔助抗癌治療之參考。

In Taiwan, cancer ranks the first of the ten leading causes of death. The statistic of 2013 published by Ministry of Health and Welfare, Executive Yuan, Taiwan showed that liver cancer was the second leading cause of cancer related deaths. However, more than 90% of cancer patients die not from their primary tumors but due to the development of metastasis. Therefore, how to prevent liver cancer and reduce cancer metastasis is a big issue in Taiwan. Previous researches demonstrated that the ethyl acetate extract from Antrodia cinnamomea suppresses the invasive potential of human hepatoma cells. The main active ingredients of Antrodia cinnamomea is ergostane-type triterpenoids, and the content of antcin K is the highest. Thus, the hypothesis of this study is that antcin K could suppresses the metastatic potential of human hepatoma cells. And the objective is that evaluating the anti-metastatic activity and mechanisms of antcin K, which is purified from the fruiting body of basswood cultivated Antrodia cinnamomea on human liver cancer cell line Hep 3B. The results show that adhesion, migration and invasion of Hep 3B cells were effectively inhibited by antcin K within 24 hours. In addition, antcin K not only reduced the protein expression and activity of MMP-2 and MMP-9, but also down-regulated vimentin, and up-regulated E-cadherin in Hep 3B cells. In depth investigation for the molecular mechanisms, revealed that antcin K could reduce the protein expression of integrin β1, β3, α5 and αv, and suppress phosphorylation of FAK, Src, PI3K, AKT, MEK, ERK and JNK. These results suggested that antcin K is able to inhibit the metastasis of human hepatoma cells through suppression of integrin-mediated adhesion, migration, and invasion. Coupled with these findings, antcin K has the good potential to reduce the risk of liver cancer metastasis.

致謝 I
摘要 II
ABSTRACT III
縮寫對照表 IV
圖次 XI
表次 XIV
第一章 文獻回顧 1
第一節 癌症 1
一、 癌症之流行病學 1
二、 癌症之簡介 1
三、 癌症的形成與發展 2
四、 癌症的特徵 3
第二節 肝臟 4
一、 肝臟之簡介 4
二、 肝臟的生理功能 5
第三節 肝癌 7
一、 肝癌之流行病學 7
二、 肝癌之簡介 8
三、 肝癌的危險因子 8
四、 肝癌的致病機轉 10
五、 肝癌的分期 11
六、 肝癌的預防與治療 13
第四節 轉移（Metastasis） 15
一、 癌症轉移的嚴重性 15
二、 癌細胞轉移的定義 15
三、 癌細胞轉移的過程 15
第五節 上皮─間質轉化（EMT） 16
一、 上皮─間質轉化與轉移 16
二、 上皮─間質轉化的過程 17
三、 上皮細胞指標蛋白─E-cadherin 19
四、 間質細胞指標蛋白─vimentin 19
第六節 侵犯（Invasion） 20
一、 癌細胞侵犯與轉移 20
二、 癌細胞侵犯的過程 20
三、 基質金屬蛋白&;#37238;（MMPs） 20
第七節 整合素（Integrin） 22
一、 細胞黏附分子（cell adhesion molecule, CAMs） 22
二、 整合素（Integrin） 23
三、 Integrin與轉移 24
第八節 Focal adhesion kinase（FAK） 26
一、 Focal adhesion kinase（FAK） 26
二、 FAK與轉移 27
三、 FAK的活化 27
四、 FAK的下游訊息傳遞途徑 27
第九節 牛樟芝（Antrodia cinnamomea） 29
一、 牛樟芝之簡介 29
二、 牛樟芝之生物學分類 30
三、 牛樟芝之命名 30
四、 牛樟芝人工栽培方法 30
五、 牛樟芝之活性成分 32
六、 牛樟芝之生理功效 39
第二章 假說與目的 43
第一節 實驗假說 43
第二節 實驗目的 43
第三章 研究架構 45
一、 Antcin K對人類肝癌細胞Hep 3B之毒性測試與安全性評估 45
二、 Antcin K對人類肝癌細胞Hep 3B之抗轉移活性與機制分析 46
第四章 材料與方法 47
第一節 化學藥品與試劑 47
第二節 實驗儀器 49
一、 細胞培養用儀器 49
二、 樣品分析用儀器 50
第三節 樣品與細胞株 50
一、 樣品 50
二、 細胞株 51
第四節 實驗方法 52
一、 細胞株之培養、解凍、繼代及保存 52
二、 細胞存活率分析（Cell viability analysis） 54
三、 細胞-人工基質黏附試驗（Cell-matrigel adhesion assay） 55
四、 細胞缺口癒合試驗（Wound healing assay） 57
五、 細胞移行能力試驗（Cell migration assay） 59
六、 細胞侵犯能力試驗（Matrigel invasion assay） 61
七、 明膠蛋白酵素電泳法（Gelatin zymography） 62
八、 西方墨點法（Western blotting） 65
九、 資料統計分析 69
第五章 實驗結果與討論 70
第一節 Antcin K對Hep 3B細胞之毒性測試與安全性評估 70
一、 Antcin K對Hep 3B細胞之細胞存活率試驗─MTT assay 70
二、 Antcin K對Hep 3B細胞之PCNA蛋白質表現量之影響 70
三、 Antcin K對肝細胞的安全性評估 71
四、 本節討論 71
第二節 Antcin K對Hep 3B細胞之抗轉移活性評估 72
一、 Antcin K對Hep 3B細胞的細胞-基質黏附作用之影響 72
二、 Antcin K對Hep 3B細胞爬行能力之影響 72
三、 Antcin K對Hep 3B細胞移行能力之影響 73
四、 Antcin K對Hep 3B細胞侵犯能力之影響 73
五、 Antcin K對Hep 3B細胞分泌MMP-2及MMP-9活性之影響 74
六、 Antcin K對Hep 3B細胞中MMP-2及MMP-9表現之影響 74
七、 Antcin K對Hep 3B細胞中EMT指標蛋白表現之影響 75
八、 本節討論 75
第三節 Antcin K對Hep 3B細胞之抗轉移機制探討 76
一、 Antcin K對Hep 3B細胞中Integrin蛋白質表現之影響 76
二、 Antcin K對Hep 3B細胞中FAK與Src蛋白質表現之影響 76
三、 Antcin K對Hep 3B細胞中PI3K/AKT蛋白質表現之影響 77
四、 Antcin K對Hep 3B細胞中MEK/ERK蛋白質表現之影響 77
五、 Antcin K對Hep 3B細胞中JNK蛋白質表現之影響 77
六、 本節討論 78
第四節 綜合討論 79
一、 Antcin K與zhankuic acid A（antcin B）之抗轉移機制比較 79
二、 Antcin K與牛樟芝子實體萃取物之抗轉移機制比較 80
第六章 結論 82
第七章 未來研究 84
一、 Antcin K之後續抗肝癌之研究 84
二、 牛樟芝子實體中麥角甾烷型三&;#33820;類之後續抗轉移之研究 84
第八章 實驗圖表 86
第九章 參考文獻 103

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