臺灣博碩士論文加值系統

牛樟芝（Antrodia cinnamomea; Syn. Antrodia camphorata or Taiwanofungus camphorata），為寄生於牛樟樹之台灣特有藥用真菌。許多研究已證實牛樟芝具有抗癌、抗發炎、降血壓、抗氧化等活性；此外，牛樟芝之菌絲體與子實體中含有特殊芳香環化合物，並已證實此類化合物具有抗癌潛力。由於癌細胞之轉移，是造成癌症病患死亡主因之一，而在癌細胞轉移之過程中，其細胞狀態、型態、貼附及移動能力皆會產生變化。其中，基質金屬蛋白酶（Matrix metalloproteinase, MMPs）扮演分解細胞外基質（Extracellular matrix, ECM）之最重要角色。當癌細胞發生了上皮-基質轉換（Epithelial-mesenchymal transition, EMT），就能輕易轉移原發器官至其他組織。因此，本研究以固態培養牛樟芝菌絲體之乙醇萃取物，分離出特有之芳香環化合物成分-利得1號（LE01, 即2,3,5-Trimethoxy-4-cresol），分析其對肺癌細胞株(A549)之轉移研究，並探討LE01在肺癌細胞之調控機制。首先，以MTT實驗分析不同濃度之LE01 (0、5、10、20、40 μM )對肺癌細胞存活率之影響，發現LE01在所有實驗濃度條件下對A549肺癌細胞株皆不具有毒殺作用，且存活率皆大於90%以上。此外，利用傷口癒合（Wound healing assay）及癌細胞遷徙抑制實驗（Transwell migration assay），來評估LE01在抗癌細胞轉移之影響，結果發現LE01在濃度40M下對A549肺癌細胞株遷移能力具有明顯之抑制效果，且呈現濃度正相關性。在浸潤實驗（Transwell invasion assay）中亦發現，濃度在40 M之LE01對細胞浸潤的情況有抑制效果，並隨LE01濃度之增加而增加。而細胞貼附（Cell adhesion assay）實驗中發現，LE01在濃度40 M處理8小時後，可有效減少70%之肺癌細胞貼附能力。進一步探討癌轉移高度相關酵素活性，包括基質金屬蛋白酶（MMPs）及尿激酶型纖溶酶原激活物（uPA），發現濃度40 M之LE01分別對MMP-2、MMP-9、uPA之酵素活性抑制率，最高達52%、35%及15%；同時利用西方墨點法，分析癌惡化轉移及侵襲之蛋白表現，發現在高濃度(40M)之LE01對上皮细胞鈣黏蛋白（E-cadherin）中之蛋白激酶B（Akt），最高抑制可達300%之抑制率；對基質金屬蛋白酶抑制劑（TIMP-1），則增加40%之表現。綜合以上試驗結果，LE01確實能經由調控上皮-基質轉換的訊息路徑，達到抗肺癌轉移之作用，未來具有做為抗肺癌轉移之藥物潛力。

Antrodia cinnamomea (Syn. Antrodia camphorata or Taiwanofungus camphorata) is a precious and unique edible fungus originated from the forest of Taiwan. In this study, an anti-metastatic compound, 2,3,5-trimethoxy-4-cresol (LE01) was isolated from the solid-state cultured mycelium of A. cinnamomea. According to the results obtained from cell wound healing, cell migration and invasion assays, LE01 suppressed effectively on movement, migration and invasion of lung cancer cell lines at the dosage in different concentrations (0、5、10、20、40 μM) without toxicity on A549 cells. Furthermore, LE01 reduced major protein expressions of MMP-2, MMP-9, Akt until 52%、35% and 300%, respectively. In addition, TIMP-1 protein expression, which is known to regulate cell adhesion, migration and invasion, was also enhanced over 40% by LE01 at the dosage of 40 μM. Therefore, these results were showed that LE01 suppressed effectively on movement, migration and invasion of lung cancer cells, and achieved the effect of anti-cancer metastasis.

Abstract i
摘要 ii
目錄 iv
圖目錄 vii
表目錄 viii
縮寫表 ix
第一章 前言 1
第一節 研究背景 1
第二節 研究動機與目的 1
第二章 文獻回顧 3
第一節 牛樟芝 3
壹、牛樟芝之分類地位 3
貳、樟芝的生長方式 3
參、樟芝的外觀特性 3
肆、樟芝的藥理活性 4
伍、樟芝的成分研究 5
陸、樟芝與靈芝的比較 7
柒、樟芝環狀化合物Leader 1介紹 8
第二節 肺癌（Lung cancer）簡介 8
壹、肺之功能及肺癌 8
貳、肺癌的分期(Stages) 9
參、罹患肺癌的危險因子（Risks） 9
肆、肺癌的診斷(Diagnosis) 11
伍、肺癌的治療(Therapy) 12
第三節 癌症轉移（Metastasis） 13
壹、癌化作用（Carcinogenesis） 13
貳、癌細胞的轉移作用（Metastasis） 13
第四節 基質金屬蛋白酶介紹（MMPs） 16
壹、基質金屬水解蛋白酵素（Matrix Metalloproteinases，MMPs） 16
貳、MMPs之抑制劑 17
參、MMPs的表現和調控 17
肆、纖維蛋白溶酶原活化物（Plasminogen activator；PA) 18
第五節 上皮基質轉換(EMT)作用 19
壹、上皮基質轉換(EMT)介紹 19
貳、上皮基質轉換(EMT)與肺癌轉移的關係 21
第三章 材料與方法 22
第一節 研究架構 22
第二節 實驗器材與藥品 23
壹、實驗抗體 23
貳、實驗綜合藥品 23
參、實驗綜合耗材 24
肆、實驗儀器 25
第三節 試驗方法 27
壹、樟芝固態培養菌絲體 27
貳、細胞培養（Cell culture） 28
參、細胞存活率分析（Cell viability） 31
肆、細胞傷口癒合實驗（Wound healing assay） 31
伍、細胞爬行試驗（Transwell migration assay） 31
陸、細胞浸潤實驗（Transwell invasion assay） 32
柒、細胞黏附實驗（Cell adhesion assay） 33
捌、基質金屬蛋白酶MMPs活性分析（Gelatin assay） 34
玖、uPA 活性分析（Casein assay） 36
拾、西方墨點法（Western blot） 37
第四章 結果 44
壹、Leader 1 對A549 細胞株之存活率 44
貳、Leader 1對A549 細胞株遷徙之抑制作用 45
参、觀察Leader 1對於細胞中MMPs及uPA活性表現的影響 50
肆、西方墨點法分析癌細胞惡化轉移及侵襲之蛋白表現 52
第五章 討論 54
第六章 參考文獻 57

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