臺灣博碩士論文加值系統

　當腫瘤生長至1~2 mm3的體積大小時，需要有新生的血管來供足夠的氧氣及養分給腫瘤快速生長，因此抑制血管新生便可阻斷腫瘤的生長。牛樟芝為台灣特有的真菌，已有許多研究指出牛樟芝萃取物能抑制腫瘤的生長及抑制血管新生。實驗室先前研究證實牛樟芝子實體萃取物 Fra-10能有效抑制血管新生，於本實驗比較 HPLC 圖譜並篩選出能有效抑制 HUVEC 生長的牛樟芝子實體萃取物。結果顯示FK4 能有效抑制 HUVEC 生長，再將 FK4 經半製備 HPLC 分離純化出 K4-2，對 HUVEC 明顯抑制生長， IC50 為 9 μg/ml。第三部分細胞遷移能力測試結果，隨著 K4-2 的濃度升高，抑制細胞遷移及生長的情形越明顯。明膠酶譜法測試結果顯示， K4-2 能抑制因 PMA 誘導 HUVEC 分泌MMP-9 蛋白，處理 PMA 及不同濃度 K4-2 皆會抑制 MMP-2 蛋白表現。微管形成能力測試結果顯示， K4-2 能抑制因 PMA 誘導 HUVEC 所形成的微管。第四部份為探討 K4-2 抑制血管新生路徑分析， K4-2 透過抑制磷酸化 VEGFR-2 蛋白表現，進而抑制下游磷酸化蛋白 Akt, PLCγ1 及 p44/42 MAPK 表現，進而抑制 HUVEC 細胞生長。 K4-2 亦會抑制 VEGFR-2 下游磷酸化蛋白Src family, FAK, p38 MAPK 表現，進而抑制細胞遷移能力。這些結果證實 K4-2會透過 VEGFR-2的訊息傳遞路徑抑制血管新生。由上述結果可知， K4-2 具有成為血管新生抑制劑的潛力。

　When the tumour grows to the volume size around 1-2 mm3, it generally requires to develop new blood vessels for supplying adequate oxygen and nutrients to the rapid growth of tumor, thus inhibiting tumor angiogenesis can block the tumor growth. Antrodia cinamomea (AC) is a unique parasitic fungus on the old camphor tree trunk cavity of the endemic species Cinnamomum kanehirae of Taiwan. Recently, many researches have indicated that extracts from AC can inhibit tumor growth and tumor angiogenesis. Previous study in our laboratory had confirmed the fruiting body’s extract Fra-10 could inhibit angiogenesis. In this study, we compared the HPLC chromatograms and screening out the new extracts which had effectively inhibit on HUVEC cell viability. The results of MTT assay showed that the FK4 fraction could effectively inhibit the growth of HUVEC cell than other tested fractions. K4-2 was further purified form FK4 fraction by using semi-preparative HPLC column, which had more significant inhibition (IC50 9 μg/ml) to the growth of HUVEC cell. The third part of this study showed that when the increase in the concentration of K4-2, the inhibition of cell migration and growth became more significantly. Zymography test results showed that K4-2 could suppress the secretion of PMA-induced MMP-9 protein level in HUVEC cell, meanwhile, MMP-2 expression was down-regulated under the treatment of PMA and K4-2. The results of tube formation assay showed that K4-2 inhibited PMA-induced microtubule. The fourth part of this study was to investigate the mechanism of K4-2 on angiogenesis signaling pathway, the results showed that K4-2 inhibited p-VEGFR-2 protein expression, and inhibited downstream p-Akt, p-PLCγ1 and p-p44/42 MAPK protein expression, resulting in the inhibition of HUVEC cell growth. K4-2 also inhibited p-VEGFR-2 downstream p-Src family, p-FAK and p-p38 MAPK protein expression, and influenced HUVEC cell migration. These results indicated that K4-2 inhibit angiogenesis through VEGFR-2 signaling pathway. Therefore, our results suggest that K4-2 might have a great potential to develop as an angiogenesis inhibitor.

中文摘要 I
Abstract III
目錄 V
圖目錄 X
附錄目錄 XII
第一章、前言 1
第二章、文獻回顧 3
一、 血管新生 3
（一） 血管新生作用 (angiogenesis) 3
（二） 腫瘤與血管新生 3
（三） 血管新生調控因子 4
二、 基質金屬蛋白酶 (Matrix metalloproteinase, MMP) 5
三、 牛樟芝 8
（一） 牛樟芝植物學分類 8
（二） 牛樟芝生長特徵 9
（三） 牛樟芝一般成分組成 10
（四） 牛樟芝活性成分 10
（五） 牛樟芝藥理活性 11
A. 抗腫瘤 11
B. 抗發炎 13
C. 抗氧化 14
D. 抗病毒 14
E. 抑制血管新生 15
第三章、研究動機與目的 17
第四章、實驗架構 19
第五章、材料與方法 21
一、 牛樟芝子實體樣本製備 21
（一） 牛樟芝子實體萃取 21
A. 利用管柱層析法分離牛樟芝子實體萃取物 21
B. 利用半製備 HPLC 分離子實體萃取物 22
二、 人類臍靜脈內皮細胞株 HUVEC 培養 22
（一） 細胞培養液製備 22
（二） 細胞型態觀察與培養繼代 23
（三） 冷凍細胞凍管與細胞活化 24
三、 細胞存活率測試 25
四、 遷移能力測試 (Wound healing assay) 25
五、 微管形成能力測試 (Tube formation assay) 26
六、 明膠酶譜法 (Gelatinase zymography) 26
（一） 蛋白質收取與定量 26
（二） 蛋白質電泳 26
（三） 呈色 27
七、 西方墨點法 (Western blot assay) 27
（一） 蛋白質萃取 27
（二） 蛋白質定量 28
（三） 蛋白質電泳及轉漬 28
（四） 免疫化學呈色 29
八、 統計分析 29
第六章、實驗結果 31
一、 牛樟芝子實體乙醇萃取物管柱層析 31
（一） 牛樟芝子實體萃取 31
（二） 管柱層析 31
二、 半製備 HPLC 純化 K4-2 31
三、 細胞存活率測試 31
（一） 溶劑乙醇對 HUVEC 細胞株之細胞存活率影響 32
（二） 牛樟芝子實體萃取物 FK1~ FK6 對 HUVEC 細胞株存活率影響 32
（三） 牛樟芝子實體萃取物 FK3-1-1, FK3-1-2, FK3-2-1 及 FK3-2-2 對 HUVEC 細胞株存活率影響 32
（四） 半製備 HPLC 純化分離物 K4-1, K4-2 對 HUVEC 細胞株存活率影響 33
（五） 溶劑乙醇對於正常細胞株 H184B5F5/M10 存活率影響………………………………………………………………...33
（六） 半製備 HPLC 純化分離物 K4-2 對 正常細胞株 H184B5F5/M10 存活率影響 33
四、 K4-2 對 HUVEC細胞株遷移能力影響 34
五、 K4-2 對 HUVEC 細胞株微管形成能力影響 34
六、 K4-2 對 HUVEC細胞株 MMP-9 及 MMP-2 蛋白影響 ………………………………………………………………35
七、 K4-2 對 HUVEC 細胞株血管新生路徑蛋白表現影響 35
（一） 磷酸化 Akt (Ser473) 蛋白表現 35
（二） 磷酸化 FAK (Tyr397) 蛋白表現 36
（三） 磷酸化 PLCγ1 (Ser1248) 蛋白表現 36
（四） 磷酸化 p38 MAPK (Thr180/Tyr182)蛋白表現 36
（五） 磷酸化p44/42 MAPK (Erk1/2) (Thr202/Tyr204) 蛋白表現…...........................................................................................37
（六） 磷酸化 Src Family (Tyr416) 蛋白表現 37
（七） 磷酸化VEGF Receptor 2 (Tyr1175) 蛋白表現 38
第七章、討論 59
第八章、結論 65
第九章、未來展望 67
第十章、參考文獻 69

Armstrong, L.C., and P. Bornstein. 2003. Thrombospondins 1 and 2 function as inhibitors of angiogenesis. Matrix Biology. 22:63-71.

Ausprunk, D.H., and J. Folkman. 1977. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvascular research. 14:53-65.

Bürgermeister, J., D.H. Paper, H. Vogl, R.J. Linhardt, and G. Franz. 2002. LaPSvS1, a (1→ 3)-β-galactan sulfate and its effect on angiogenesis in vivo and in vitro. Carbohydrate research. 337:1459-1466.

Björklund, M., and E. Koivunen. 2005. Gelatinase-mediated migration and invasion of cancer cells. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 1755:37-69.

Brew, K., D. Dinakarpandian, and H. Nagase. 2000. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1477:267-283.

Brown, L., M. Detmar, K. Claffey, J. Nagy, D. Feng, A. Dvorak, and H. Dvorak. 1997. Vascular permeability factor/vascular endothelial growth factor: a multifunctional angiogenic cytokine. In Regulation of angiogenesis. Springer. 233-269.

Chang, T., and W. Chou. 1995. Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan. Mycological Research. 99:756-758.

Cheng, J.-J., N.-K. Huang, T.-T. Chang, D. Ling Wang, and M.-K. Lu. 2005. Study for anti-angiogenic activities of polysaccharides isolated from Antrodia cinnamomea in endothelial cells. Life Sciences. 76:3029-3042.

Dimmeler, S., E. Dernbach, and A.M. Zeiher. 2000. Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS letters. 477:258-262.

Folkman, J. 1971. Tumor angiogenesis: therapeutic implications. N Engl j Med. 285:1182-1186.

Folkman, J. 1995. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature medicine. 1:27-30.

Folkman, J. 2004. Endogenous angiogenesis inhibitors. Apmis. 112:496-507.

Folkman, J. 2006. Antiangiogenesis in cancer therapy—endostatin and its mechanisms of action. Experimental Cell Research. 312:594-607.

Geback, T., M.M. Schulz, P. Koumoutsakos, and M. Detmar. 2009. TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays. BioTechniques. 46:265-274.

Geethangili, M., and Y.-M. Tzeng. 2011. Review of pharmacological effects of Antrodia camphorata and its bioactive compounds. Evidence-Based Complementary and Alternative Medicine. 2011.

Genersch, E., K. Hayeß, Y. Neuenfeld, and H. Haller. 2000. Sustained ERK phosphorylation is necessary but not sufficient for MMP-9 regulation in endothelial cells: involvement of Ras-dependent and-independent pathways. Journal of Cell Science. 113:4319-4330.

Hsiao, G., M.-Y. Shen, K.-H. Lin, M.-H. Lan, L.-Y. Wu, D.-S. Chou, C.-H. Lin, C.-H. Su, and J.-R. Sheu. 2003. Antioxidative and hepatoprotective effects of Antrodia camphorata extract. Journal of Agricultural and Food Chemistry. 51:3302-3308.

Ilan, N., S. Mahooti, and J.A. Madri. 1998. Distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis. Journal of Cell Science. 111:3621-3631.

Kroll, J., and J. Waltenberger. 1997. The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. Journal of Biological Chemistry. 272:32521-32527.

Kumar, V.B., T.-C. Yuan, J.-W. Liou, C.-J. Yang, P.-J. Sung, and C.-F. Weng. 2011. Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 707:42-52.

Lamalice, L., F. Le Boeuf, and J. Huot. 2007. Endothelial cell migration during angiogenesis. Circulation research. 100:782-794.

Lee, I., R.L. Huang, C.T. Chen, H.C. Chen, W.C. Hsu, and M.K. Lu. 2002. Antrodia camphorata polysaccharides exhibit anti‐hepatitis B virus effects. FEMS Microbiology Letters. 209:63-67.

McCawley, L.J., and L.M. Matrisian. 2000. Matrix metalloproteinases: multifunctional contributors to tumor progression. Molecular medicine today. 6:149-156.

McDonnell, S., M. Morgan, and C. Lynch. 1999. Role of matrix metalloproteinases in normal and disease processes. Biochemical Society Transactions (I 999). 27.

Montesano, R., and L. Orci. 1985. Tumor-promoting phorbol esters induce angiogenesis in vitro. Cell. 42:469-477.

Moser, T.L., M.S. Stack, I. Asplin, J.J. Enghild, P. Højrup, L. Everitt, S. Hubchak, H.W. Schnaper, and S.V. Pizzo. 1999.
Angiostatin binds ATP synthase on the surface of human endothelial cells. Proceedings of the National Academy of Sciences. 96:2811-2816.

Nagase, H., and J.F. Woessner. 1999. Matrix metalloproteinases. Journal of Biological Chemistry. 274:21491-21494.

O'Reilly, M.S., L. Holmgren, Y. Shing, C. Chen, R.A. Rosenthal, M. Moses, W.S. Lane, Y. Cao, E.H. Sage, and J. Folkman. 1994. Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma. Cell. 79:315-328.

Parsons, S., S. Watson, P. Brown, H. Collins, and R. Steele. 1997. Matrix metalloproteinases. British Journal of Surgery. 84:160-166.

Schnaper, H.W., D.S. Grant, W.G. Stetler‐Stevenson, R. Fridman, G. D'Orazi, A.N. Murphy, R.E. Bird, M. Hoythya, T.R. Fuerst, and D.L. French. 1993. Type IV collagenase (s) and TIMPs modulate endothelial cell morphogenesis in vitro. Journal of cellular physiology. 156:235-246.

Shu, C.-H., and M.-Y. Lung. 2008. Effect of culture pH on the antioxidant properties of Antrodia camphorata in submerged culture. Journal of the Chinese Institute of Chemical Engineers. 39:1-8.

Snoek, P., and J.W. Von den Hoff. 2005. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.
Spinale, F.G. 2007. Myocardial matrix remodeling and the matrix metalloproteinases: influence on cardiac form and function. Physiological reviews. 87:1285-1342.

Still, K., C.N. Robson, P. Autzen, M.C. Robinson, and F.C. Hamdy. 2000. Localization and quantification of mRNA for matrix metalloproteinase‐2 (MMP‐2) and tissue inhibitor of matrix metalloproteinase‐2 (TIMP‐2) in human benign and malignant prostatic tissue. The Prostate. 42:18-25.

Wang, M., T. Wang, S. Liu, D. Yoshida, and A. Teramoto. 2003. The expression of matrix metalloproteinase-2 and -9 in human gliomas of different pathological grades. Brain tumor pathology. 20:65-72.

Weidner, N. 1993. Tumor angiogenesis: review of current applications in tumor prognostication. In Seminars in diagnostic pathology. Vol. 10. 302-313.

Wen, C.L., C.C. Chang, S.S. Huang, C.L. Kuo, S.L. Hsu, J.S. Deng, and G.J. Huang. 2011. Anti-inflammatory effects of methanol extract of Antrodia cinnamomea mycelia both in vitro and in vivo. J Ethnopharmacol. 137:575-584.

Wu, S.-H., and L. Ryvarden. 1997. Antrodia camphorata (" niu-chang-chih"), new combination of a medicinal fungus in Taiwan. BOTANICAL BULLETIN-ACADEMIA SINICA TAIPEI. 38:273-276.

Wu, S.-H., Z.-H. Yu, Y.-C. Dai, C.-T. Chen, C.-H. Su, L.-C. Chen, W.-C. Hsu, and G.-Y. Hwang. 2004. Taiwanofungus, a polypore new genus. Fungal Sci. 19:109-116.

Xia, C., Q. Meng, L.-Z. Liu, Y. Rojanasakul, X.-R. Wang, and B.-H. Jiang. 2007. Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer research. 67:10823-10830.

Zang, M., and C. Su. 1990. Ganoderma comphoratum, a new taxon in genus Ganoderma from Taiwan, China. Acta Botanica Yunnanica. 12:395-396.

吳威翰. 2012. 牛樟芝子實體萃取物誘導肺癌細胞進行細胞凋亡之研究. 國立東華大學生物技術研究所碩士論文.

吳書慧. 2013. 特定牛樟芝子實體純化物對人類胃癌細胞株 AGS 之細胞凋亡機制探討. 國立東華大學生物技術研究所碩士論文.

李雨薇. 2007. 牛樟芝菌絲體醱酵液對血管新生抑制作用及其機制之探討. 長庚大學生化與生醫工程研究所碩士論文.

高翊愷. 2011. 牛樟芝子實體萃取物對於人類肝癌細胞株 HepG2之細胞凋亡機制探討. 國立東華大學生物技術研究所碩士論文.

高曉薇. 1991. 台灣靈芝屬新種樟芝之三萜類成分研究. 台北醫學院天然物醫學研究所碩士論文.

張力中. 2014. 牛樟芝子實體純化物antcin A在肝癌異種移植模型中誘導腫瘤細胞凋亡機制探討. 國立東華大學生物技術研究所碩士論文.

許勝傑, 陳清農, 陳勁初. 2000. 樟芝宿主專一性之探討. 台灣農業化學與食品科學. 38:533-539.

陳勁初, 林文鑫, 陳清農, 許勝傑, 黃仕政, 陳炎鍊. 2001. 台灣特有真菌-樟芝菌絲體之開發. Fungal Science. 16:7-22.

陳啓楨, 蘇慶華, 藍明煌. 2001. 樟芝固體栽培及其生物活性之研究. Fungal Science. 16:65-72.

彭梓涵. 2012. 特定牛樟芝子實體純化物對人類卵巢癌細胞株PA-1、OVCAR-3之影響. 國立東華大學生物技術研究所碩士論文.

羅仁傑. 2009. 牛樟芝中抑制血管新生活性成分之研究. 國立東華大學生物技術研究所碩士論文.

羅宇浩. 2014. 特定牛樟芝子實體純化物對人類前列腺癌細胞在動物實驗內之細胞凋亡機制探討. 國立東華大學生物技術研究所碩士論文.

蘇慶華. 2004. 健康的守護神: 國寶樟芝. 愛克思文化出版.