臺灣博碩士論文加值系統

大腸直腸癌 (colorectal cancer, CRC) 是世界上主要死因之ㄧ。 2011年台灣行政院衛生署公佈統計結果顯示，大腸直腸癌為所有癌症之發生增加率第一位。其通常使用的治療方法是手術或化療，手術治療是最主要的方式，大約有將近半數大腸直腸癌的患者可以由手術與抗癌藥物的合併治療達到治癒，但是癌症的轉移通常會造成治療的失敗。因此探討以天然食材開發抗癌物質為目前科學家主要研究方向之一。鴻喜菇 (Hypsizygus marmoreus) 是近年來流行的可食性菇類，具有高蛋白質、低熱量及低脂肪等特點，並具有多種生理活性如抗腫瘤和抗真菌等活性。本研究使用冷鹽水萃取鴻喜菇，經40-80％ 硫酸銨沉澱製備樣品。經連續鹽梯度分離的第三個區分物命名為HM-3。HM-3 (50 μg/ mL) 與人類大腸直腸癌HCT116細胞作用12和24小時後，可抑制HCT116細胞生長達57.2%及81.9%。在傷口癒合試驗(wound healing assay)中，觀察到HM-3可抑制HCT116細胞移行且具有時間效應。在Transwell試驗中，經濃度10 - 40 μg/ mL HM-3處理HCT116 達48小時後也可抑制HCT116細胞的侵入性。接著進一步利用明膠酶譜測定HCT116細胞培養液中基質金屬蛋白酶（matrix metalloproteinases, MMPs）的活性, 結果顯示隨著HM-3刺激濃度的升高可降低MMP-2和MMP-9 的活性。以西方墨點法探討抑制轉移的可能路徑，當HM-3刺激濃度從0增加至40 μg/ mL，mTOR的表現量下降，其下游轉移相關的蛋白質Twist和N-cadherin的表現量也減少，而E- cadherin表現量增加。因此，本實驗推測HM-3可能透過mTOR訊息傳遞路徑而達到抑制HCT116細胞轉移的能力。

Colorectal cancer (CRC) is one of the top causes of death in the world. The statistics of 2011 published by Department of Health, Executive Yuan, Taiwan showed that CRC has taken the first place of rate of increase in all kinds of cancers. The normal treatments are surgery or chemotherapies with the former being the major one. About half of patients with colorectal cancer can be cured by surgery and multimodal treatment, while metastasis is the most important cause for cure failure. Therefore, development of effective chemotherapeutic agents from natural food is rewarding. Hypsizygus marmoreus (HM) is one of the most popular dietary mushrooms that contain high protein, low calorie and low fat with antitumor activitiy.
In the present study, 40~80% ammonium sulfate precipitates of cold-salt water extracts from HM were prepared. DEAE-Sepharose CL-6B ion exchange chromatography was used, eluted by 0-1 M NaCl continuous gradient, and the fraction 3 isolated by continuous gradient is named HM-3. The growth inhibition of HCT116 cells treated with 50 μg/ mL HM-3 for 12 and 24 hrs were 57.2% and 81.9%, respectively. In wound healing assay, the inhibition of HCT116 cells migration was observed, and the results are time-dependent. The invasion of HCT116 was inhibited by 10 to 40 μg/ mL HM-3 treated for 48 hrs in transwell assay. Furthermore, the activity of matrix metalloproteinases (MMPs) was detected by gelatin zymography assay. The results showed that increase in the concentration of HM-3 could decrease the activity of MMP-2 and MMP-9. The inhibition of metastasis pathway was analyzed by the western blotting. The expression of mTOR, Twist and N-cadherin proteins was decreased while the expression of E-cadherin was increased by treating with 0 – 40 μg/ mL HM-3. In conclusion, we propose that HM-3 is able to inhibit the migration and invasion of colorectal cancer cells through mTOR signaling pathways.

摘要 I
Abstract II
第一章、前言 1
第二章、 文獻回顧 3
第一節、癌症 3
一、癌症簡介 3
二、大腸直腸癌 3
(一)、大腸直腸癌成因 3
(二)、大腸直腸癌的分期 5
(三)、大腸直腸癌的治療方式 7
第二節、食用菇 13
一、食用菇簡介 13
二、鴻喜菇 14
第三節、癌細胞之移行、侵入及轉移 17
一、移行與侵入 17
二、轉移 17
第四節 基質金屬蛋白酶 19
一、基質金屬蛋白酶 19
二、活性調控 20
第五節 表皮-間質轉換 (epithelial mesenchymal transition, EMT) 22
一、EMT的發生 22
二、細胞黏附蛋白 ( E-cadherin、N-cadherin) 22
三、EMT轉錄因子 (Twist) 24
四、雷帕黴素標靶蛋白 (mammalian target of Rapamycin, mTOR) 24
第三章 研究動機 26
第四章 實驗架構 27
第五章 材料與方法 28
第一節、實驗材料 28
一、原料來源 28
二、實驗細胞 28
三、藥品與試劑 28
四、藥品配置 31
五、實驗儀器 34
第二節、實驗方法 35
一、鴻喜菇蛋白質粗萃物製備 35
二、40-80%飽和度硫酸銨沉澱之蛋白質粗萃物之陰離子交換樹脂分離 35
三、人類大腸直腸癌HCT116細胞之活化、培養及保存 36
四、生長抑制測試-MTT assay 37
五、創傷癒合試驗 (Wound healing assay) 38
六、細胞移行試驗 (Migration assay) 39
七、細胞侵入試驗 (Invasion assay) 40
八、細胞收集與蛋白質萃取 41
九、明膠酶譜 (Gelatin Zymography) 41
十、蛋白質濃度定量 42
十一、SDS-PAGE 43
十二、Coomassie Brilliant Blue 染色及退染 44
十三、西方墨點法 (Western blotting) 44
十四、統計分析方法 46
第六章 實驗結果與討論 46
一、鴻喜菇蛋白質粗萃物製備 46
二、40-80%飽和度硫酸銨沉澱之鴻喜菇蛋白質粗萃物之陰離子交換樹脂連續鹽梯度分離 47
三、鴻喜菇醣蛋白質HM-3之SDS-PAGE檢定 47
四、鴻喜菇醣蛋白質HM-3對HCT116細胞之生長抑制測試-MTT assay 48
五、創傷癒合試驗 (Wound healing assay) 48
六、細胞侵入試驗 (Invasion assay) 49
七、細胞移行試驗 (Migration assay) 50
八、明膠酶譜法 (Gelatin Zymography) 51
九、以西方墨點法探討蛋白質HM-3抑制HCT116細胞移行與侵入機制 51
第七章 結論 53
第八章 參考文獻. 54

水野卓、川合正允，賴慶亮譯。1997。菇類的化學、生化學。國立編譯館，台北。
今關六也、本鄉次雄。1957。原色日本菌類圖鑑。保育社，東京。
王怡凱、王緯書、周月卿、劉俐婷。2008。轉移性大腸癌病例之用藥探討。醫院藥學，25，128-132。
行政院衛生署。2013。行政院衛生署衛生統計資料網。
徐麗嵐。2002。以柳松菇與鴻喜菇誘導人類白血病細胞(U937)分化及對Balb/c鼠皮下移植CT26腫瘤之抑制效果。國立台灣大學食品科技研究所碩士論文。
徐素琴。2007。台灣山豆根根部粗抽物誘導人類肝癌細胞 (Hep3B)細胞週期停滯、細胞凋亡與抑制轉移之分子機轉。中國醫藥大學中國藥學研究所博士論文。
高明見。2007。免疫系統與細胞免疫療法。遠東聯合診所期刊。
陳光耀。1972。癌病淺說。科學月刊，3，13-14。科學月刊社，台北。
陳勁初、黃仕政。2000。菇菌類機能性食品之開發。生物產業 ，11(3)，164-172。
陳品玲、林美良、林佳靜、張鶴齡、羅琦、馮清淳、鄭春秋。2008。成人
內外科護理上冊，第四版，p535。華杏，台北。
陳秋慧。2005。癌症化學治療及護理。癌症護理學，華杏，台北。
陳建男。2010。玉皇菇中一種新免疫調節蛋白之純化與其生理活性之研究。國立台灣大學食品科技研究所博士論文。
張雅筑。2012。鴻喜菇蛋白質對人類肝癌細胞 (HepG2) 生長抑制及細胞週期停滯之研究。國立台灣大學食品科技研究所博士論文。
郭志鎰。2001。大腸直腸癌致癌機轉之研究。中山醫學大學生物化學研究
所碩士論文。
趙大中。2008。轉移性大腸直腸癌的標靶治療。臨床醫學，6，99-104。
葉怡柔、林水木、許峯旗、林逸祥、簡素玉。2009。大腸直腸癌之藥物治療。藥學雜誌，101，93-95。
鄭武飛。1989。免疫细胞膜分子（二）。醫學免疫學。北京，人民衛生出版社。
歐馨婷。2001。多種蔬菜抑制人類白血病細胞U937增殖與誘導其分化之探討。國立台灣大學食品科技研究所碩士論文。
蔡慧思、魏聖修、劉俊坤、許健成。2010。基質金屬蛋白酶-2,9 與腫瘤細胞轉移
之重要性。醫檢會報，25，48-55。
簡佳怡。2005。鴻喜菇蛋白質對小鼠大腸癌CT26細胞生長抑制機制及對Balb/c鼠之該腫瘤轉移效果。國立台灣大學食品科技研究所碩士論文。
蔣軍廣、王麗華、譚偉麗、陳秋生、帖永新、翟建華、趙娜。2008。N-cadherin在非小細胞肺癌組織中的表達及臨床意義。中國腫瘤臨床，35，96-99。
謝銘鈞。2004。大腸癌當代醫學，3，953-959。
潘函君。2008。探討人類乳癌細胞中 P0 及 GCIP 蛋白和 tumor progression 之關係。國立成功大學生物科技研究所碩士論文。
謝佳瑛、宋慧蒂、陳芳婷、陳彥仰。2007。大腸直腸癌的標靶治療。長庚
藥學學報，14，4-9。
癌症問答。2007。http://www.cancerquest.org/index.cfm?page=399&lang=tchinese
轉移介紹。2004。http://www.oxygentimerelease.com/A/Disorders/p24.htm
醫源世界。2007。惡性腫瘤的生成。
Abdalla, E. K.; Vauthey, J. N.; Ellis, L. M.; Ellis, V.; Pollock, R.; Broglio, K. R.; Hess, K.; Curley, S. A. Recurrence and outcomes following hepatic resection, radiofrequency ablation, and combined resection/ablation for colorectal liver metastases. Ann Surg. 2004, 239, 818-825.
Abid, M. R.; Yi, X.; Yano, K.; Shih, S. C.; Aird, W. C. Vascular endocan is preferentially expressed in tumor endothelium. Microvasc Res. 2006, 72, 136-145.
American cancer society: Colorectal cancer facts & figures. American cancer society. Atlanta. 2011.
Bozzuyo, G.; Molinri, A. Molecular aspects of tumor cell migration and invasion. Ann. Ist. Super. Sanita. 2010, 46, 66-80.
Cheung, P. C. Plasma and hepatic cholesterol levels and fecal neutral sterol excretion are altered in hamsters fed straw mushroom diets. J. Nutr. 1998, 222, 687-688.
Chang, J. S.; Son, J. K.; Li, G.; Oh, E. J.; Kim, J. Y.; Park, S. H.; Bae, J. T.; Kim, H. J.; Lee, I. S.; Kim, O. M.; Kozukue, N.; Han, J. S.; Hirose, M.; Lee, K. R. Inhibition of cell cycle progression on HepG2 cells by hypsiziprenol A9, isolated from Hypsizigus marmoreus. Cancer Lett. 2004, 212, 7-14.
Dolley, C. M.; McEwan, J. R.; Henney, A. M. Matrix metalloproteinases and vardiovascular disease. Circ Res. 1995, 77, 863-868.
Deryugina, E. I.; Quigley, J. P. Matrix metalloproteinases and tumor metastasis. Cancer Metastasis Rev. 2006, 25, 9-14.
Fishman, D. A.; Liu, Y.; Ellerbroek, S. M.; Stack, M. S. Lysophosphatidic acid promotes matrix metalloproteinase (MMP) activation and MMP-dependent invasion in ovarian cancer cells. Cancer Res. 2001, 61 , 3194-3199.
Gory, S.; Dalmon, J.; Prandini, M. H.; Kortulewski, T.; Huber, P. Requirement of a GT box (Sp1 site) and two Ets binding sites for vascular endothelial cadherin gene transcription. J Biol Chem. 1998, 273, 6750-6755.
Gulhati, P.; Bowen, K. A.; Jianyu , Stevens, P. D.; Rychahou, P. G.; Chen, M.; Lee , E. Y.; Weiss, H. L.; O’Connor, K. L Gao, T.; Evers, B. M. mTORC1 and mTORC2 regulate EMT, motility, and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res. 2011, 71 , 3246-3256.
Hikino, H.; Ishiyama, M.; Suzuki, Y.; Konno, C. Mechanisms of hypoglycemic activity of Ganderan B: a glycan of Gandoderma lucidum fruit bodies. Planta Med. 1989, 55, 423-428.
Huhtala, P.; Tuuttila, A.; Chow, L.T.; Lohi, J. Complete structure of the human
gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells. J. Biol. Chem. 1991, 266, 16485–16490.
Howell, G. M.; Humphrey, L. E.; Awwad, R. A. Aberrant regulation of transforming growth factor-alpha during the establishment of growth arrest and quiescence of growth factor independent cells. J Biol Chem. 1998, 273, 9214.
Harvey, L.; Arnold, B.; Lawrence, Z.; Paul, M.; David, B.; James, D. Molecular cell biology, 4rd edition; 1999, p.1060.
Hendrix, M. J.; Seftor, E. A.; Hess, A. R.; Seftor, R. E. Molecular plasticity of human melanoma cells. Oncogene. 2003, 22, 3070-3075.
Haijuan,Y.; Derek, G.; Rudge, J.; Bhamini, V.; Hyo, Y.; Nikola, P. mTOR kinase structure, mechanism and regulation. Nature. 2013, 497,217-224.
Iwamuro, Y.; Aoki, M.; Mikami, Y. J. Ferment Technol. 1985, 63, 61-66.
Ikekawa, T.; Saitoh, H.; Feng, W.; Zhang, H.; Li, L.; Matsuzawa, T. Antitumor activity of Hypsizigus marmoreus. I. Antitumor activity of extracts and polysaccharides. Chem. Pharm. Bull. 1992, 40, 1954-1957.
Iozzo, R.V.; Cohen, I. Altered proteoglycan gene expression and the tumor stroma. Experientia. 1993, 49, 447-455.
Iwatsuki, M.; Mimori, K.; Yokobori, T.; Ishi, H.; Beppu , T.; Nakamori, S.; Baba, H.; Mori, M. Epithelial–mesenchymal transition in cancer development and its clinical significance. Cancer Sci. 2009, 101 , 293-299.
Jiang, D.; Yang, H.; Willson, J. K. Autocrine transforming growth factor alpha provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states. J Biol Chem. 1998, 273, 31471.
Joyce, E. R. Matrix metalloproteinases and angiogenesis. J. Cell. Mol. Med. 2005, 9, 267-285.
Kazutaka, T.; Yutaka, A.; Shoji, O.; Takashi, M.; Mamoru, I. Isolation of novel collagen-binding protein from the mushroom, Hypsizigus marmoreus, which inhibits the Lewis Lung carcinoma cell adhesion to Type IV collagen. J Biol Chem. 1995, 270(4), 1481-1484.
Kelloff, G. J.; Schilsky, R. L.; Alberts, D. S.; Day, R. W.; Guyton, K. Z.; Pearce, H. L.; Peck, J. C.; Phillips, R .; Sigman, C. C. Colorectal adenomas: a prototype for the use of surrogate end points in the development of cancer prevention drugs. Clin Cancer Res. 2004, 10, 3908-3918.
Lieu, C.W.; Lee, S. S.; Wang, S.Y. The effect of Ganoderma lucidum on induction of differentiation in leukemic U937 cells. Anticancer Res. 1992, 12, 1211-1216.
Lam, S. K.; Ng, T. B. Hypsin, a novel thermostable ribosome-inactivating protein with antifungal and antiproliferative activities from fruiting bodies of the edible mushroom Hypsizigus marmoreus. Biochem. Biophys. Res. Commun. 2001, 285, 1071-1075.
Mohammed, F. F.; Smookler, D. S.; Khokha, R. Metalloproteinases, inflammation, and rheumatoid arthritis. Ann Rhum Dis. 2003, 62, 43-47.
Milli, A.; Perego, P.; Beretta, G. L.; Corvo, A.; Righetti, P. G.; Carenini, N.; Corna, E.; Zuco, V.; Zunino, F.; Cecconi, D. Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells. J Proteome Res. 2011, 10, 1191-1207.
Nagase, H.; Woessner, J. F. Matrix metalloproteinases. J Biol Chem. 1999, 274,
21491–21494.
Nakai.; R, Masui.; H, Horio.; H, Ohtsuru.; M. Effect of maitake (Grifola frondosa) water extract on inhibition of adipocyteconversion of C3H10T1/2B2C1 cells. J Nutrition SciVitaminol. 1999, 45, 385-389.
Nguyen, M.; Arkell, J.; Jackson, C. J. Human endothelial gelatinases and angiogenesis. Int J Biochem Cell Biol. 2001, 33, 960-970.
Obrocea, F.; Sajin, M.; Marinescu, E.; Stoica, D. Colorectal cancer and the 7th revision of the TNM staging system: review of changes and suggestions for uniform pathologic reporting. Rom J Morphol Embryol. 2011, 52, 537-544.
Peng, B. Suppression of human ovarian SKOV-3 cancer cell growth by Duchesnea phenolic fraction is associated with cell cycle arrest and apoptosis. Gynecol Oncol. 2008, 108, 173-181.
Roy, R.; Zhang, B.; Moses, M. A. Making the cut: proteasemediated regulation of
angiogenesis. Exp Cell Res. 2006, 312, 608-622.
Rychahou, P. G.; Jackson, L. N.; Silva, S. R.; Rajaraman, S.; Evers, B. M. Targeted molecular therapy of the PI3K pathway: therapeutic significance of PI3K subunit targeting in colorectal carcinoma. Ann Surg. 2006, 243, 833-842.
Sopata, J. A latent gelatin specific proteinase of human leucocytes and its
activation. Biochim. Biophys. Acta. 1979, 571, 305–312.
Sichel, G.; Corsaro, C.; Scalia, M.; Bilio, A. J.; Bonomo, R. P. In vitro scavenger activity of some flavonoids and melanins against O2. Free Radical Biology and Medicine . 1991, 11, 1-8.
Saitoh, H.; Feng, W. J.; Matsuzawa, T.; Ikekawa, T. Antitumor activity of Hypsizigus marmoreus. II. Preventive effect against lung metastasis of Lewis lung carcinoma. Yakugaku Zasshi. 1997, 117, 1006-1010.
Shoji, Y.; Isemura, M.; Muto, H.; Isemura, S.; Aoyagi, Y. Isolation of a 41-kda protein with cell adhesion activity for animal tumor cells from the mushroom hypsizigus marmoreus by affinity chromatography with type iv collagen immobilized on agarose. Biosci Biotechnol Biochem. 2000, 64(4), 775-780.
Sternlicht, M.; Coussens, L.; Vu, T. H.; Werb, Z. Biology and regulation of the matrix metalloproteinases. In Cancer drug discovery and development: Matrix metalloproteinase inhibitors in cancer therap . Humana Press Inc.2000,1-37.
Sawhney, R. S.; Zhou, G. H.; Humphrey, L. E. Differences in sensitivity of biological functions mediated by epidermal growth factor receptor activation with respect to endogenous and exogenous ligands. J Biol Chem. 2002, 277, 75-80.
Suyama, K.; Shapiro, I.; Guttman, M. A signaling pathway leading to metastasis is controlled by N-cadherin and FGF receptor. Cancer Cell. 2002, 24, 301-314.
Stefanidakis, M.; Koivunen, E. Cell-surface association between matrix
metalloproteinases and integrins: role of the complex in leukocyte migration and cancerprogression. Blood. 2006, 108, 1441-1450.
Sehgal, S. N. Its discovery, biological properties, and mechanism of action. Transplant Proc. 2003, 35, 7-14.
Tam, S. C.; Yip, K. P.; Fung, K. P.; Chang, S.T. Hypotensive and renal effects of an extract of the edible mushroom Pleurotus sajor-caju. Life Sci. 1986, 38, 1155-1161.
Vu, T. H.; Werb, Z. Matrix metalloproteinases: effectors of development and normal physiology. Gene. 2000, 14, 2123-2133.
Wang, J.; Sun, L.; Myeroff, L.; Demonstration that mutation of the type II transforming growth factor beta receptor inactivates its tumor suppressor activity in replication error-positive colon carcinoma cells. J Biol Chem.1995; 270, 22044.
Wang, H. X.; Ooi, V. E.; Ng, T. B.; Chiu, K. W.; Chang, S. T. Hypotensive and vasorelaxing activities of a lectin from the edible mushroom Tricholoma mongolicum. Pharm Toxicol. 1996, 79, 318-323.
Wang, J. J.; Lee, J. Y.; Chen, Y. C.; Chern, Y. T.; Chi, C.W. The antitumor effect of a novel differentiation inducer, 2, 2-Bis (4-(4-amino-3-hydroxyphenoxy) phenyl) adamantane (DPA), in combinatory therapy on human colon cancer. Int J Oncol . 2006, 28, 1003-1012.
Wallerand, H.; Robert, G.; Pasticier, G.. The epithelial-mesenchymal transition-inducing factor TWIST is an attractive target in advanced and/or metastatic bladder and prostate cancers. Urol Oncol Semin Invest. 2010, 28, 473-479.
Yamamoto, Y.; Shirono, H.; Kono, K.; Ohashi, Y. Immunopotentiating activity of the water-soluble lignin rich fraction prepared from LEM-the extract of the solid culture medium of Lentinus edodes mycelia. Biosci Biotechnol Biochem. 1997, 61, 1909-1912.
Vartio, T.; Vaheri, A. A gelatin-binding 70,000-dalton glycoprotein synthesized
distinctly from fibronectin by normal and malignant adherent cells. J. Biol. Chem. 1981, 256, 13085–13090.
Van, P. E.; Dubois, B.; Nelissen, I. Rudd, P.M. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9). Crit. Rev.Biochem. Mol. Biol. 2002, 37, 375–536.
Vihinen, P.; Kahari, V. M.; Matrix metalloproteinases in cancer: Prognostic markers and therapeutic targets. Int J Cancer. 2002, 99, 157-166.