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研究生:楊喬筑
研究生(外文):Chian-ju Yang
論文名稱:以細胞培養模式評估固態培養牛樟芝菌絲體萃取物之抗肝癌生物活性及其機制
論文名稱(外文):Assessment of solid-state culture of Antrodia cinnamomun mycelia extracts on antitumor effect and mechanisms in cultured hepatoma cells
指導教授:龔瑞林龔瑞林引用關係
指導教授(外文):Zwe-Ling Kong
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:85
中文關鍵詞:牛樟芝肝癌細胞凋亡
外文關鍵詞:CASPASETRAIL
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中文摘要
牛樟芝(Polyporaceac,Aphyllophorales)在台灣是眾所周知的一種傳統中藥,並且已被用來做為各種疾病的治療,包含食物藥物中毒、皮膚癬、肝癌等。目前已知牛樟芝具有抗氧化、抗發炎、保肝及抗癌的能力,本論文旨在評估固態培養的牛樟芝菌絲體乙醇萃取物之活性,並探討其抑制肝癌細胞增生的情形及誘導毒殺肝癌細胞的機制。本文中先利用人類肝癌細胞Hep G2為實驗對象,以測其細胞存活率的方式,評估出固態培養的牛樟芝菌絲體乙醇萃取物有效區間,並挑選出有效區間J101進行細胞毒殺機制的探討。以J101處理HepG2 48 小時後,測其IC50的值為0.5�慊/ml。經由測其DNA片段化現象得知J101經由apoptosis造成細胞死亡,更進一步發現隨著處理的劑量增加其Caspase 3有被活化之現象。接著使用TRAIL和另一活性區間EACPS共同處理細胞探討肝癌細胞株的TRAIL抗性,發現其具有促進Hep G2 和Hep 3B 之APOPTOSIS,推論EACPS 與TRAIL之間的協同活性會促進細胞死亡。由本論文實驗結果推論,固態培養的牛樟芝菌絲體乙醇萃取物具有造成肝癌細胞Hep G2細胞凋亡的現象,其作用機制可能是活化下游的caspase 3,而造成Hep G2 APOPTOSIS。
關鍵字:牛樟芝、肝癌、細胞凋亡、CASPASE、TRAIL
Abstract
Antrodia cinnamomun (Polyporaceac, Aphyllophorales) is a well-known Chinese herbal medicine in Taiwan. It has been using for the treatment of a variety of diseases, such as food and drug intoxication, diarrhea, abdominal pain, hypertension, itchy skin and liver cancer. At present, it has been known that Antrodia cinnamomun can play the role on anti-oxidation, anti-inflammation, hepatoprotective and antitumor. The purpose of this study is to evaluate the effect of ethanolic extracts of mycelia cultured from the biologically active fraction of A. cinnamomun on the inhibition of cell viability and its cytotoxic induced mechanism in hepatoma cell line (Hep G2). In the research, Hep G2 is uesed for testing cell viability to evaluate the active fraction of ethanolic extracts of mycelia cultured from the biologically active fraction of A. cinnamomun. And the active fraction J101 is picked out to probe the mechanism of apoptosis. The IC50 value was 0.5�慊/ml when being treated HepG2 cell line with J101 for 24 and 48hrs. By testing DNA fragment , it was known that J101 leads the cell death was by apoptosis. We nearly explore to the mechanism of apoptosis that the more the dose of caspase 3 enzymes activity that behave. The present result implies that ethanolic extract from A. cinnamomun mycelia may lead to inhibit proliferation and induces apoptosis pathway on hepatoma HepG2 cell line. To further exploration of the TRAIL resistance in hepatoma cell line, we process cells with TRAIL and EACPS. It is founded that induce Hep G2 and Hep 3B apoptosis. Therefore, according to the result of this study, we estimate that ethanolic extracts of mycelia cultured from A. cinnamomun of biologically active compounds can contribute to apoptosis of Hep G2 which is triggered by activating caspase 3 enzyme .
Key words: Antrodia cinnamomun, liver cancer, apoptosis, caspase, TRAIL.
目錄
目錄 i
圖表目錄 iv
附錄 vi
中文摘要 vii
Abstract viii
名詞縮寫 ix
壹、緒論 1
貳、文獻回顧 3
一、 牛樟芝的簡介 3
(一)名稱及分類: 3
(二)型態 4
(三)寄生情況 5
(四)樟芝的成分分析 5
1. 化學成分分析 5
2. 生理活性成分分析 6
(五)樟芝深層培養及其在生理機能之研究 7
1. 深層培養方法 7
2. 安全性與毒性之相關研究 9
3. 抗氧化功能之相關研究 9
4. 保肝功能之相關研究 10
5. 免疫蛋白之純化與調節免疫活性 10
6. 預防心血管疾病及降血糖之相關研究 11
7. 調節免疫力及抗腫瘤功能之相關研究 11
二、肝癌與細胞凋亡(Apoptosis) 13
(一) 肝癌 13
(二)肝癌的治療方法 13
(三) APOPTOSIS 14
(四) 細胞死亡模式分析 14
三、細胞凋亡的調控途徑 15
(一) 停止細胞週期(cell cycle) (Alberts et al., 1998): 15
(二) 細胞凋亡的執行者—caspase 16
(三) 死亡受體訊息傳遞 (Signal transduction induced death receptor pathway ) 17
(四) 粒線體調控途徑 (Mitochondrial associated pathway) 17
(五) 活性氧(Reactive Oxygen Species; ROS)的氧化傷害 19
四、TRAIL (Tumor necrosis factor related apoptosis–inducing ligand) 抗性 19
參、實驗設計 22
肆、材料與方法 23
一、實驗材料 23
1. 細胞株 23
2. 樟芝菌絲體來源 23
二、藥品與試劑 23
三、設備 26
四、實驗方法 27
1. 樟芝菌絲體乙醇萃取液的製備 27
(1) EAC (Extract of Antrodia cinnamomun) 製備 27
(2) EACHW (Extract Antrodia cinnamomun from Hot Water)製備 27
(3) EACPS(Extract of Antrodia cinnamomun Polysaccharides fraction)製備 27
(4) EACPS1 (Extract of Antrodia cinnamomun Polysaccharides fraction 1)製備 28
(5) EACPS2 ( Extract of Antrodia cinnamomun Polysaccharides fraction 2)製備 28
(6) EACIN (Extract Antrodia cinnamomun insoluble fraction)製備 28
(7) EACIN層進一步分離 28
(8) J101層的分離 29
(9) TLC之呈色反應 30
2. 細胞的培養與凍結 30
(1) 細胞培養 30
(2) 細胞凍結 31
3. 細胞存活率試驗(MTT assay) 31
4. LDH KIT 31
5. DNA 片段化之測定 (DNA Fragmentation Test) 32
6. Caspase 8,Caspase 3酵素活性之測定 33
7. 粒線體膜電位ΔΨm 之測定 (Palmeira et al., 1996) 33
陸、總結 42
柒、參考文獻 43
捌、圖表 55
玖、附錄 81
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