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標題: | 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 Anti-migration and Anti-invasion Mechanisms of Glyco-protein HM-3 from Hypsizygus marmoreus against Human Colorectal HCT116 Cancer Cells |
作者: | Tzu-Yu Li 李紫瑜 |
指導教授: | 吳瑞碧 |
共同指導教授: | 馬嘉軉 |
關鍵字: | 傷口癒合試驗,移行,侵入,基質金屬蛋白酶, wound healing assay,invasion,migration,matrix metalloproteinases,MMPs, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61595 |
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顯示於系所單位: | 食品科技研究所 |
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