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系統識別號 U0002-2507200715545500
中文論文名稱 松杉靈芝甲醇萃取物誘發人類表皮癌細胞細胞凋亡
英文論文名稱 Ganoderma tsugae methanol extracts could induce apoptosis in human epidermoid A431 cell
校院名稱 淡江大學
系所名稱(中) 化學學系碩士班
系所名稱(英) Department of Chemistry
學年度 95
學期 2
出版年 96
研究生中文姓名 黃逸民
研究生英文姓名 Ii-Ming Huang
學號 694171090
學位類別 碩士
語文別 中文
口試日期 2007-07-07
論文頁數 51頁
口試委員 指導教授-莊子超
委員-王文奇
委員-高銘欽
委員-莊子超
中文關鍵字 癌症  細胞凋亡  EGFR  松杉靈芝 
英文關鍵字 cancer  apoptosis  EGFR,  Ganoderma tsugae 
學科別分類 學科別自然科學化學
中文摘要 在亞洲國家中,靈芝是一種用來藥用和養生的菌類。近年來文獻指出靈芝具有免疫修飾 (immunomodulatory) 和抗腫瘤的能力 (anti-tumor) 。另一方面而言,表皮生長因子受體 (Epidermal Growth Factor Receptor, EGFR) 是在許多癌症中具有過度表現或是基因突變的一種原致癌基因,其中發生在皮膚癌、大腸癌、乳癌、腦癌和子宮頸癌等等。我們發現在松杉靈芝甲醇萃取高濃度 (1 mg/ml) 下,對人類表皮癌 A431 細胞不只有明顯的細胞毒性,且也會誘發細胞凋亡。本論文中,我們證實松杉靈芝甲醇萃取物誘發EGFR過度表現的人類表皮癌A431細胞凋亡可能的路徑。實驗結果中發現,松杉靈芝甲醇萃取物能夠有效的降低 A431 細胞 EGFR 蛋白表現並且也發現也能降低Akt活性。這顯示松杉靈芝甲醇萃取物誘發A431細胞凋亡和降低 EGFR 具有緊密的關係。更近一步探討,也發現靈芝甲醇萃取物能夠誘發粒腺體釋放 cytochrome c 並活化下游 caspase 3 和抑制 PARP 活性,最後也發現松杉靈芝甲醇萃取物能夠降低A431的抗細胞凋亡蛋白 (Bcl-2, Bcl-xL) 表現。因此松杉靈芝甲醇萃取物對於EGFR過度表現A431 細胞不只降低 EGFR 蛋白表現也抑制其下游訊號傳遞,而抑制其增生作用,最後誘發由 caspase 所引起的細胞凋亡。
英文摘要 Ganoderma (Lingzhi), an oriental fungus, has been widely used as a medical drug or health-promotion product in Asian countries. Lingzhi have been demonstrated to possess immunomodulatory and anti-tumor activities. On the other hand, the Epidermal Growth Factor Receptor (EGFR) is frequently amplified, overexpressed, or mutated in many cancers, including skin, colon, lung, breast, head and neck cancer. We have investigated, at high doses (>1 mg/ml), the methanol extracts from the Ganoderma tsugae
(G. tsugae) could be cytotoxic through induction of apoptosis. In the present study, we examined the effect of G. tsugae extract on apoptotic biochemical events in EGFR-overexpressing human epidermoid carcinoma A431 cells. G. tsugae extracts treatment could down-regulate the EGFR protein level and cause the de-phosphorylation/inactivation of constitutively active AKT. This data suggest that G. tsugae extracts could induce apoptosis might through downregulation of EGFR in A431 cells. Furthermore,
G. tsugae extracts also could induce release of cytochrome c accompanied by activation of caspase-3 and PARP cleavage. Finally, treatment of A431 cells with G. tsugae extracts could down-regulate the expression of inhibitors of apoptosis protein, such as Bcl-2 and Bcl-xL. Taken together, our finding suggest that G. tsugae extracts could down-regulate EGFR protein level and its downstream signaling pathway in EGFR-overexpressing human epidermoid carcinoma A431 cells leading to the inhibition of
proliferationand induction of caspase-dependent apoptosis.
論文目次 目錄
論文提要內容…………………………………………………………………...Ⅰ
Abstract..………………………………………………………………………...Ⅱ
目錄……………………………………………………………………………...Ⅲ
圖表目錄………………………………………………………………………...Ⅵ
附錄目錄………………………………………………………………………...Ⅶ
名詞縮寫………………………………………………………………………...Ⅷ
第一章、序論
第一節、細胞凋亡…………………………………………………………01
壹、細胞凋亡概論…………………………………………………….01
貳、細胞凋亡的調控…………………………………………………02
第二節、上皮生長因子接受體 (EGFR) ………………………………...04
壹、EGFR 概論………………………………………………………04
貳、針對EGFR 的治療方法…………………………………………05
第三節、靈芝………………………………………………………….......06
壹、靈芝概論…………………………………………………………06
貳、靈芝的分類、分部及其外型構造……………………..…………06
叁、靈芝的化學組成和生理功用…………………………………….07
肆、松杉靈芝…………………………………………………………10
第四節、實驗源起與目的…………………………………………………12
第二章、材料與方法…………………………………………………………….13
第一節、實驗材料及相關儀器……………………………………………13
壹、細胞株……………………………………………………….......13
貳、抗體……………………………………………………………...13
叁、化學藥品及試劑………………………………………………...13
肆、主要儀器及器材………………………………………………...14
第二節、實驗方法………………………………………………………...15
壹、松杉靈芝萃取物的製備……………………………………..….15
貳、西方點墨法……………………………………………………...16
叁、分析釋放 cytochrome c量……………………......……..…..….17
第三章、結果………………………………………….……………….……….19
壹、松杉靈芝甲醇萃取物對人類上皮細胞A431 之型態觀察
……………………………………………………………….…..19
貳、松杉靈芝萃取物對於EGFR 蛋白表現量……..…………….....20
叁、松杉靈芝萃取物對於cyt c 蛋白表現量的影響………..……...21
肆、松杉靈芝萃取物對於Bcl-2 和Bcl-xL 蛋白表現量的影響……..22
伍、松杉靈芝萃取物對於PARP 和pro-caspase 3 蛋白表現量
…………………………………………………………….……….23
第四章、討論………………………………………………………….………....25
第五章、結論…………………………………………………………………….27
第六章、參考文獻……………………………………………………………….28
圖………………………………………………………………………………...34
表………………………………………………………………………………...39
附錄……………………………………………………………………………...48
圖表目錄
圖1: 松杉靈芝甲醇萃取物處理A431 細胞型態觀察………………………..34
圖2: 松杉靈芝萃取物對於EGFR蛋白表現量………………………………..35
圖3: 松杉靈芝萃取物對於cyt c 蛋白表現量………………………………....36
圖4: 松杉靈芝萃取物對於Bcl-2 和Bcl-xL蛋白表現量………………………37
圖5: 松杉靈芝萃取物對於PARP 和pro-caspase 3 蛋白表現量………….…38
表1. 《神農本草經》六種靈芝………………………………………………..39
表2. 台灣常見八種野生靈芝………………………………………………...40
表3. 靈芝在2004 年前所發表過的功效……………………………….……42
表4. 細胞株基本資料……………………………………………………...…44
表5. 西方點墨法之抗體資料………………………………………………...45
表6. 化學藥品及試劑(chemicals and reagents)…………………………...46
附錄目錄
附錄1 細胞培養使用之緩衝溶液………………………………………..…48
附錄2 西方點墨法使用之化學試劑………………………………….…….49
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