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研究生:李紫瑜
研究生(外文):Tzu-Yu Li
論文名稱:鴻喜菇醣蛋白質HM-3抑制人類大腸直腸癌細胞HCT116移行與侵入機制之研究
論文名稱(外文):Anti-migration and Anti-invasion Mechanisms of Glyco-protein HM-3 from Hypsizygus marmoreus against Human Colorectal HCT116 Cancer Cells
指導教授:吳瑞碧
口試委員:沈賜川廖慧芬
口試日期:2013-06-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:105
中文關鍵詞:傷口癒合試驗移行侵入基質金屬蛋白酶
外文關鍵詞:wound healing assayinvasionmigrationmatrix metalloproteinasesMMPs
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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



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