牛樟芝是台灣特有的藥用真菌之一，是極為珍貴的藥材，民間相傳其具有治療食物中毒、腹痛、癌症及解毒之功效，目前在抗癌藥劑的研究上也廣為盛行。但其僅少量生長於台灣特有的牛樟樹上，且子實體生長速度極為緩慢。由於野生牛樟芝的來源不易取得，因此利用液態培養來進行生產，以獲得大量的牛樟芝菌絲體及其代謝產物為目前重要之研究課題。
本研究之目的在探討液態培養的菌絲體醱酵液是否具有抑制血管新生作用。研究中我們檢驗菌絲體醱酵液對不同的內皮細胞株(SVEC與EA. hy 926)的細胞毒性(cytotoxicity)，及對其遷移(migration)、脈管形成(tube formation)的抑制能力；在細胞分子機制方面測試其對小鼠大腸癌細胞株(CT26) 、小鼠肝癌細胞株(1MEA.7R)及人類肝癌細胞株(HA22T)血管內皮生長因子(VEGF)與缺氧誘導因子(HIF-1α)表達的抑制能力；最後在雞胚絨毛尿囊膜上進行初步體內(in vivo)試驗。實驗結果顯示牛樟芝菌絲體醱酵液確實具有抑制血管新生的作用，其不但能抑制血管內皮細胞增生、遷移及脈管形成並能抑制多種癌細胞表現血管內皮生長因子，另外，雞胚絨毛尿囊膜實驗上，也發現牛樟芝菌絲體醱酵液對雞胚的血管生長具有明顯抑制效果。

Antrodia camphorata is a Taiwan-unique medicinal fungus that been widely used for the treatment of food intoxication, detoxification and also has effective antitumor activity. Generally, it is becoming difficult to find A. camphorata in the forest because of its slow growing fruiting body. We develop submerged culture to gain deal of A. camphorata mycelia for its metabolite.
The main purposes of this study were to characterize A. camphorata mycelia fermentation broth (AC-MFB), and investigate its potential anti-angiogenic effect. The anti-angiogenic capability of AC-MFB was investigated by cell proliferation, migration, and tube formation assay using endothelial cells. Several essential molecular mechanisms of anti-angiogenesis were also investigated, including regulation on vascular endothelial growth factor (VEGF) expression and its inhibition of hypoxia induced factor-1α (HIF-1α) under DFO induced hypoxic condition in mouse adenocarcinoma (CT26), hepatoma (1MEA.7R) and human hepatoma cell lines (HA22T). We also used chorioallantoic membrane (CAM) assay as in vivo model. The results showed decreased endothelial cells viability by AC-MFB in both dose and time-dependent manners. AC-MFB also inhibited the migration and tube formation in endothelial cells. Moreover AC-MFB reduced nuclear localization of HIF-1α and VEGF expression in CT26, HA22T and 1MEA.7R in the presence of DFO stimulation. In CAM assay, the result showed that AC-MFB could inhibition the development of blood vessel. In conclusion, AC-MFB not only suppressed endothelial viability, migration and tube formation, but also had inhibition effects on HIF-1α and VEGF.

目錄
指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
授權書………………………………………………………….……..…iii誌謝……………………………………………………...…………....…iv
中文摘要……………………………………………………………....…v
英文摘要……………………………………………………..…….……vi
目錄………………………………………………………...………...…vii圖目錄………………………………………………………………..... xii表目錄………………………………………………………….…….…xv
第一章 緒論……………………………………………………………...1
1.1研究動機…………………………………………………………...1
1.2研究目的…………………………………………………………...2
1.3研究架構………………………………………………………….2第二章 文獻回顧………………………………………………………...3
2.1牛樟芝簡介.………………………………………………………..3
2.2牛樟芝生理活性成分.……………………………………..………4
2.3牛樟芝的療效……….……………………………………………..4
2.4牛樟芝培養方式及其比較……….....……………………….…….6
2.5惡性腫瘤………………………………………………...…………7
2.5.1腫瘤成長………………………………………………………8
2.5.2腫瘤轉移…………………………………………………...….9
2.6血管新生作用…………………………………………………..….9
2.6.1血管新生作用與癌症………………………………………..10
2.7 調節血管新生作用之相關因子…………………………………11
2.7.1缺氧…………………………………………………………..11
2.7.2缺氧誘導因子………………………………………………..12
2.7.2.1 缺氧誘導因子簡介…………………………………..…12
2.7.2.2缺氧誘導因子調控機制………………………………...12
2.7.2.3缺氧誘導因子-1α與癌症………………………….……14
2.7.3血管內皮生長因子(VEGF)….………………………………15
2.8抗血管新生療法與癌症治療…………………………………….15
2.9太平洋紫杉醇簡介……………………………………………….16
2.9.1太平洋紫杉醇結構……………………………………….….17
2.9.2 Taxol作用機轉………………………………………………17
2.10 不同藥物合併使用之功效……………………………………..18第三章 實驗材料與方法…………………………………………….…19
3.1 牛樟芝菌絲體醱酵液製備…………………………………...….19
3.1.1 牛樟芝菌絲體培養………………………………………….19
3.1.2實驗藥品與儀器………………………………………......…20
3.1.3菌絲體醱酵液製備……………….………………………….22
3.1.4菌絲體醱酵液多醣體濃度測定……………………………..22
3.1.4.1多醣體之萃取…………………………………………...22
3.1.4.2多醣體濃度測定-酚硫酸法……………………………..22
3.2 動物細胞培養……………………………………………………24
3.2.1 細胞株………………………………………………….……24
3.2.2 培養基組成……………………………………………….…24
3.2.3 實驗藥品與儀器…………………………………………….25
3.2.4 細胞培養方式……………………………………………….26
3.2.5 繼代培養…………………………………………………….26
3.2.6 動物細胞解凍與保存……………………………………….26
3.3細胞毒性試驗 (MTT assay)……………………………………..27
3.3.1原理…………………………………………………..………27
3.3.2實驗藥品與儀器 …………………………………...……….28
3.3.3方法…………………………………………………..………29
3.3.4計算方式………………………………………………..……29
3.4 內皮細胞移行能力測試 (migration assay - wounding assay)….30
3.4.1實驗藥品與儀器……………………………………………..30
3.4.2方法…………………………………………………….….…30
3.4.3計算方式……………………………………………………..31
3.5內皮細胞脈管生成之試驗 (Tube formation assay)………..……31
3.5.1實驗藥品與儀器………………………………………..……31
3.5.2方法………………………………………………………..…32
3.5.2.1 Coating Matrigel Mtraix…………………………….…..32
3.5.2.2 種入細胞…………………………………………..……32
3.5.3分析方式……………………………………………………..32
3.6 偵測惡性腫瘤細胞株中血管內皮生長因子的表現量….…...…33
3.6.1 實驗藥品與儀器……………………………………...……..33
3.6.2 方法……………………………………………………….....34
3.6.2.1樣品製備………………………………………………...34
3.6.2.2蛋白質濃度測量…………………………………...……34
3.6.2.3 VEGF ELISA assay………………………………..……35
3.6.3計算方式……...……………………………………….……..35
3.7偵測惡性腫瘤細胞中HIF-1α的表現量………………….…..….36
3.7.1實驗藥品與儀器………………………………………...…...36
3.7.2方法…………………………………………………………..36
3.7.2.1樣品製備………………………………………………...36
3.7.2.2蛋白質濃度測量………………………………...………37
3.7.2.3 HIF-1α ELISA assay…………………………….………37
3.8雞胚胎絨毛尿囊膜(Chorioallantoic Membran)血管形成試驗….38
3.8.1雞蛋來源……………………………………………..………38
3.8.2培養條件…………………………………………………..…38
3.8.3方法………………………………………………………..…39
3.9動物毒性試驗………………………………………………….…39
3.9.1動物來源…………………………………………………..…39
3.9.2飼養環境………………………………………………..……40
3.9.3方法……………………………………………………..……40
3.9.3.1牛樟芝菌絲體醱酵液之急毒性試驗………………...…40
3.9.3.2牛樟芝菌絲體醱酵液之28天餵食毒性試驗……….…40
3.10統計分析……………………………………….…………....…..41第四章 結果與討論.................................................................................42
4.1 牛樟芝菌絲體醱酵液成分分析……………………………...….42
4.1.1 多醣體含量………………………………………………….42
4.1.2保存時間對多醣體含量之影響……………………………..42
4.2 牛樟芝菌絲體醱酵液對細胞型態改變之影響…………..……..42
4.3 樟芝菌絲體醱酵液對細胞之毒殺能力…………………………43
4.4牛樟芝菌絲體醱酵液對內皮細胞遷移之影響………………….44
4.5 牛樟芝菌絲體醱酵液抑制內皮細胞株脈管形成之能力……....45
4.6牛樟芝菌絲體醱酵液抑制腫瘤細胞株HIF-1α之表現…………46
4.7牛樟芝菌絲體醱酵液抑制腫瘤細胞株VEGF的表現………….47
4.8雞胚胎絨毛尿囊膜血管生成試驗……………………………….48
4.9 合併藥物毒性……………………………………………….…...49
4.9.1 細胞毒性…………………………………………………….49
4.9.2 細胞遷移抑制能力….………………………………………50
4.9.3脈管形成抑制能力…………………………………………..51
4.10動物毒性試驗…………………………………………………...52 第五章 結論與未來展望……………………………………………….53 參考文獻………………………………………………………………...77

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