樟芝生長於台灣特有品種牛樟樹樹幹內，為台灣民間廣為流傳的天然藥物。一般用來當作解毒劑或治療高血壓、高血脂及抗癌。經實驗顯示樟芝富含三帖類、倍半帖類、胺基酸、蛋白質、糖類和固醇類、多醣體，具相當高的營養價值。另一方面，研究指出，從樟芝子實體體所萃取出來的取出來的固醇類物質zhankuic acid 對於小鼠血癌細胞P338具有毒殺效果。之後，又有科學家發現樟芝菌絲體水萃取物會毒殺人類血癌細胞HL-60，但對於培養的人類內皮細胞卻不具此活性。截至目前為止，對於樟芝抗腫瘤方面的研究侷限在體外實驗(in vitro)。因此，我們想要評估樟芝甲醇萃取液(ACME)在體內(in vivo)及體外(in vitro)，其整體的抗癌效果為何。
　　體外實驗方面，於一序列細胞毒殺試驗中，發現ACME對於許多癌細胞皆有毒殺效果，且具劑量正相關性。我們進一步利用流式細胞儀，以PI染色，觀察樟芝萃取液對細胞週期的影響，發現ACME會使腫瘤細胞週期停頓在Sub-G1 phase。之後，再以Hoechst 33258進行細胞核染色及DNA片段電泳分析，結果顯示ACME處理之後，癌細胞趨向程式死亡。體內實驗方面，我們發現背負ML1-4A腫瘤細胞的Balb/c小鼠腫瘤及背負LLC腫瘤細胞之C57BL/6小鼠的腫瘤皆不同層級的被ACME所抑制。在癌細胞移行(cell migration assay)實驗中，我們發現ACME對PC-3人類前列腺癌細胞移行能力具有抑制效果，進一步利用電泳酵素分析(Gelatin Zymography)發現ACME會降低PC-3分泌MMP-9但對MMP-2卻無顯著影響，推測其可能具抑制腫瘤細胞侵入的能力。關於血管新生方面，ACME可抑制BAEC（牛肺動脈內皮細胞）的移行，同時於雞胚胎絨毛膜試驗中高劑量ACME可顯著抑制雞胚胎血管新生。將ML1-4A原位腫瘤以CD31染色，發現血管無明顯被抑制。因此，推測ACME的抗腫瘤效果主要在於抑制癌細胞的生長而非血管的增生。

　　Antrodia camphorata, or ”chang-chih” in its folk name, is a tree fungus that has been used by Taiwanese indigenes for years for its anti-inflammatory activity. Several compounds have been identified from the fruiting body of Antrodia camphorate, such as triterpenoids, sesquiterpenoids, steroids, and polysaccharides. Previous researches indicated that zhankuic acid extracted from fruit body of A. camphorata exhibited cytotoxicity against P388 murine leukemia and aqueous extract of A. camphorata reduced the viability of HL-60 human leukemia cells. And yet no animal experiments and other aspects of anticancer activities of A. camphorata have been tested. My study aims at investigating the anticancer activity of a methanolic extract obtained from cultured mycelia of A. camphorate, termed ACME.
　　The extract shows markedly dose-dependent cytotoxicities against several cancer cell lines. The flow cytometry and DNA fragmentation assays demonstrate that the cytotoxicity is due to cell apoptosis. The anticancer activity of ACME was investigated further in vivo using mouse bearing transplanted tumor cells. ACME significantly inhibits the growths of tumors both in the Lewis lung carcinoma and ML1-4A hepatoma animal models. Besides, we found ACME inhibits the migration of human prostate cancer cells (PC-3) toward chemo-attractant and reduces MMP-9 but not MMP-2 secretion, suggesting ACME possesses an anti-metastatic principle. ACME also exhibits anti-angiogenesis activities in an endothelia cell migration assay and blood vessel formation in a semi in vivo chicken chorioallantonic membrane (CAM) assay. But no apparent differences are found in CD31 immunohistochemistry of ML1-4A primary tumors from either control animal or animal treated with ACME, indicating the tumor-inhibiting activity is not due to angiogenesis inhibition.
　　The active anticancer ingredient and its detailed molecular action mechanism in A. camphorate warrant further investigation.

考試合格證明
致謝
目錄 I
圖目錄 III
表目錄 IV
中文摘要 V
英文摘要 VI
縮寫表 VII
第一章 研究背景 1
第二章 實驗材料 8
第一節 細胞株 8
第二節 樟芝甲醇萃取物(ACME)之配製 8
第三節 試劑 8
第三章 實驗方法 11
第一節 細胞培養 11
第二節 離體細胞毒性 12
第三節 細胞週期之分析 14
第四節DNA片段電泳分析法 15
第五節 Hoechst 33258 細胞核染色法 16
第六節 癌細胞誘發皮下原位腫瘤模式 17
第七節 癌細胞移行測試 18
第八節 電泳酵素分析 19
第九節 內皮細胞移行試驗 20
第十節 雞胚胎絨毛膜血管新生試驗 22
第十一節 CD31組織免疫化學染色法 23
第四章 實驗結果 26
第五章 討論 34
參考文獻 38
圖表 44
作者簡歷 63

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