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研究生:徐瑋憶
研究生(外文):Wei-Yi Hsu
論文名稱:以蛋白質體及流式細胞儀探討紅麴米誘導Caco-2細胞及樟芝萃取液誘導HepG2細胞之凋亡
論文名稱(外文):Proteomic and flow cytometry analysis of apoptosis in Monascus-fermented red mold rice-treated Caco-2 cells and Antrodia cinnamomea extracts-treated HepG2 cells
指導教授:林文源林文源引用關係
指導教授(外文):Wun-Yuan Lin
學位類別:碩士
校院名稱:實踐大學
系所名稱:食品營養與保健生技研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:130
中文關鍵詞:紅麴樟芝二維電泳monacolin K
外文關鍵詞:Monascus spp.Antrodia cinnamomeatwo-dimentional electrophoresismonacolin K
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近年來癌症為十大死因之首,所以開發具防癌機能性之食品或是藥品為現今值得研究的課題。紅麴菌及樟芝都是目前應用於健康食品的真菌,紅麴菌(Monascus spp.)在中國民間應用已有數千年之久,先前的研究指出紅麴菌二次代謝產物中monacolin K、monascorubrin及ankaflavin 具有抑制腫瘤細胞增生的作用。樟芝(Antrodia cinnamomea)又稱牛樟芝是台灣特有真菌,僅生長於台灣特有之牛樟樹樹心中,其生長緩慢、數量稀少,所以價格昂貴,樟芝在台灣傳統醫藥上被視為珍貴藥材,一般民間認為具有保健及治病的功效。
研究中試驗紅麴及樟芝萃取物對人類直腸癌細胞株Caco-2及人類肝癌細胞株HepG2的影響,利用洋菜凝膠電泳、流式細胞儀並以顯微鏡觀察細胞的形態變化來偵測細胞凋亡情形。此外,應用蛋白質體分析系統分析紅麴甲醇萃取物處理Caco-2細胞及使用樟芝甲醇萃取物處理Caco-2及HepG2細胞偵測蛋白質表現。結果證實細胞處理紅麴萃取物會負調控一些蛋白包括heat shock protein 70、protein kinase C ε type、clusterin-associated protein、及兩個腫瘤抑制蛋白 (N-chimaerin和calponin-2) ,此結果顯示包含heat shock protein 70可能為紅麴萃取物誘導細胞死亡的原因。
另一方面,實驗證明使用75μM紅麴萃取物或是400 μg/ml樟芝萃取物處理Caco-2及HepG2細胞株24小時皆能有效的誘導細胞早期凋亡發生。試驗結果可以來幫助推測紅麴及樟芝萃取液如何影響腫瘤細胞的基因傳達並可能進一步研究其抑制腫瘤細胞的機制。
Cancer is the first of ten major causes of the death in recent years. The object of this study is to develop the functional health food or/and the medicines against cancer cell proliferation. Monascus species and Antrodia cinnamomea both are the fungi applied to the health food recently. Monascus-fermented red mold rice in China, has been extensively used as a folk medicine for thousands of years. Monascus secondary metabolites, including monacolin K, monascorubrin, and ankaflavin, have been reported to have an antiproliferative effect on cancer cells. A. cinnamomea is a very expensive and rare mushroom because it slowly grows on the inner heart wood wall of the Cinnamomum kanehiraae only, which is an unique tree in Taiwan with limited amount. A. cinnamomea is a traditional Taiwanese herbal medicine well known for its potential health improvement and disease prevention.
This study investigates the effects of different levels of Monascus-fermented red mold rice extract (MRE) and A. cinnamomea extract (ACE) in Caco-2 and HepG2 cells. We evaluated apoptosis action of MRE and ACE against Caco-2 and HepG2 cells by DNA laddering in agarose gels, FACScan flow cytometry, and by microscopy. Furthermore, we used proteomic approach and observed the appreciable deregulation in protein expressions after treatment with MRE in Caco-2 cell, and treatment with ACE in Caco-2 and HepG2 cells. The deregulated protein expressions, after treatment with MRE, were identified including heat shock protein 70, protein kinase C ε type, clusterin-associated protein 1, and two tumor suppressors (N-chimaerin and calponin-2). This results suggested the involvement of heat shock protein 70-mediated cytotoxicity in the Caco-2 cells treated with MRE.
In addition, the study revealed that the 75μg/ml of MRE or 400μg/ml of ACE could effectively induce early apoptosis action in Caco-2 and HepG2 cells for 24-hour exposure. The results of this study help us to get insight into the effect of MRE and ACE on the gene expressions in Caco-2 and HepG2 cells.
目錄………………………………………………………………… Ι
圖目次……………………………………………………………… Ⅲ
表目次……………………………………………………………… Ⅶ
摘要………………………………………………………………… Ⅸ
英文摘要…………………………………………………………… Ⅹ
第一章 前言……………………………………………………… 1
第二章 文獻回顧………………………………………………… 2
第一節 紅麴菌……………………………………………… 2
第二節 樟芝………………………………………………… 10
第三節 癌症………………………………………………… 16
第四節 細胞凋亡與細胞壞死……………………………… 18
第五節 蛋白質體學………………………………………… 22
第六節 研究目的…………………………………………… 26
第三章 材料與方法……………………………………………… 27
第一節 實驗材料…………………………………………… 27
第二節 實驗方法…………………………………………… 33
第三節 二維蛋白質膠體電泳分析實驗…………………… 41
第四章 結果……………………………………………………… 48
第五章 討論……………………………………………………… 53
第六章 結論……………………………………………………… 66
第七章 參考文獻………………………………………………… 116
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