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研究生:許筌翔
研究生(外文):Chiuan-Shiang Shu
論文名稱:牛樟芝甲醇萃取物誘發A549 肺腺 癌細胞自我凋亡機制之探討
論文名稱(外文):Studies on the Mechanisms of Apoptosis induced by the Methanol Extract of A.camphorate in A549 cells.
指導教授:姜泰安姜泰安引用關係林淑瑗林淑瑗引用關係
指導教授(外文):Tai-An Chiang Ph.D.Shwu-Yuann Lin Ph.D.
口試委員:姜泰安林淑瑗吳佩芬
口試委員(外文):Tai-An Chiang Ph.D.Shwu-Yuann Lin Ph.D.Pei-Fen Wu
口試日期:2014-01-13
學位類別:碩士
校院名稱:中華醫事科技大學
系所名稱:醫學檢驗生物技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:44
中文關鍵詞:A549肺癌細胞細胞凋亡caspase牛樟芝
外文關鍵詞:A549 cellapoptosiscaspaseA. camphorate
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肺癌是全世界男女性癌症死因之首,也是台灣十大癌症死因之首。台灣每天約有22位民眾死於肺癌,其中19位是因非小細胞肺癌死亡。非小細胞肺癌主要包括腺癌、鱗狀上細胞、與大細胞癌。非小細胞肺癌占85%~87%的肺癌病例。台灣的牛樟芝是一種多孔菌,在台灣已經被認為樟芝對癌症具有療效。曾有許多研究指出,牛樟芝具有對各種癌細胞的抗腫瘤及抗癌效果,也在臨床醫學上用於治療多種疾病,如肝病,腫瘤,中毒,腹痛等等。
將實驗室培養的A549肺癌細胞中,添加不同濃度(0~30 μg/ml)的牛樟芝萃取液,以細胞毒性的MTT assay、流式細胞分析儀(flowcytometry)與西方墨點法(western blot analysis)分析探討其誘發細胞自我凋亡之機制。細胞毒性MTT分析結果指出在濃度40μg/ml 已經對A549細胞產生毒性,因此不影響結果的狀況下,我們選用了15 ,20, 25,30μg/ml 的A. camphorate進行實驗。流式細胞分析儀分析結果指出,牛樟芝的濃度會上升是因為細胞apoptosis情形越來越明顯。再者,由西方墨點法之結果顯示,發現cytochrome C、Caspase-9、Caspase-3、Caspase-7、PARP蛋白質的表現量隨著牛樟芝萃取液的濃度增加而增加,在BCL-XL的protein表現量會依牛樟芝的濃度增加而減少。研究顯示,牛樟芝萃取物誘導A549細胞生長,它未來可能在抗癌藥物
中具有高度應用價值。
Backgrounds: Lung cancer is the first cause of death in men and women all over the
world. It is also the first cause of death induced by cancer in Taiwan. There are about 22 people died for lung cancer per day, where 19 of 22 is the result of non-small cell lung cancer. Non-small cell lung cancer includes Adencarcinoma,quamous cell, and large cell carcinoma, and it accounts for about 85 % of lung cancer cases. It had been known for many years that selected mushrooms are effective in fighting against cancer in stomach, esophagus, lungs and others, whereas the mechanisms of anticancer therapeutic effects of mushrooms are poorly understood. Antrodia camphorata is a cultured volvatus mushroom in Taiwan. Bioactive effects in A.camphorate have been the focus of recent research on antitumor, and anticancer activity, and also that the treatment of various diseases, such as liver disease, cancer,poisoning, abdominal pain, and apply on clinical medicine. Our previous study demonstrated that the methanol extract from A. camphorate has induction activity of apoptosis in MCF-7 breast cells. It was suggested that MEAC may be a potential chemo preventive agent on apoptosis. The aim of the present study was to investigate the mechanisms of apoptosis induced by MEAC in A549 cells.
Materials and Methods: Cytotoxicity of different concentration (0 ~ 40 μg / ml) of
MEAC has been measured by MTT cytotoxicity analysis. We further verify the mechanism of MEAC on cytotoxicity in human lung adenocarcinoma A549 cells with
flow cytometry analysis and Western blotting analysis. Results: Our data showed that
cytotoxic activity induced by MEAC in a dose-response manner. Therefore, we chose
15, 20, 25, 30μg/ml of MEAC to do the experiments without affecting the results. The result of flow cytometry analysis has been demonstrated that cell apoptosis was also significantly induced by MEAC in a dose-response pattern. Moreover, western blotting assay showed that cytochrome C, Caspase-9, Caspase-3, Caspase-7, 89 kDa PARP (poly ADP-ribose polymerase) protein expression increased with the increasing concentration of MEAC. Furthermore, BCL-XL protein expression performance will depend on the amount of increase in the reducing concentration of MEAC. Collectively, the mechanism of apoptosis induced by MEAC in A549 cells may be partially mediated through induction of Caspase protein expression and inhibition of BCL-XL protein expression. Conclusion: The study had shown that MEAC through induction of apoptosis pathway, then inhibit the growth of A549 cancer cells. So, MEAC may be a highly value product with anti-cancer drugs.
致謝..................................................................... ii
目錄..................................................................... iii
摘要..................................................................... iv
Abstract ............................................................... vi
第一章、前言............................................................... 1
1-1 肺癌介紹 ............................................................. 1
1-2 台灣牛樟芝 ............................................................ 4
1-3 細胞凋亡 .............................................................. 8
第二章、研究目的............................................................ 11
第三章、實驗材料與方法....................................................... 12
3-1 實驗材料 .............................................................. 12
3-2 實驗儀器 .............................................................. 17
3-3 實驗方法 .............................................................. 18
第四章、結果.................................................................24
4-1 牛樟芝甲醇萃取物的細胞毒性分析 ............................................. 24
4-2 牛樟芝甲醇萃取物的流式細胞分析儀(flow cytometry)測定 ................... .....25
4-3 牛樟芝甲醇萃取物的西方墨點(Western Blot)測定 ............................... 26
第五章、討論與結論............................................................ 28
參考文獻.............................................................. ..... 30
圖表附錄.............................................................. ..... 33
Fig.1.Effects of A. camphorate on A549 cell growth. ....................... 33
Fig.2. Phase-contrast micrographs of A. camphorate on A549 cell growth. ... 34
Fig.3. Effects of A. camphorate on A549 cell apoptosis measured by flow
cytometry analysis. ........................................................ 35
Fig.4.Western blot analysis of BCL-XL protein expression in A549 cells. .... 36
Fig.5.Western blot analysis of Cytochrome C protein expression in A549 cells.37
Fig.6.western blot analysis of Caspase-9 protein expression in A549 cells. . 38
Fig.7.Western blot analysis of Caspase-3 protein expression in A549 cells... 39
Fig.8.Western blot analysis of Caspase-7 protein expression in A549 cells... 40
Fig.9.western blot analysis of PARP protein expression in A549 cells. ...... 41
附錄一:Apoptosis pathway .................................................... 42
附錄二:細胞凋亡過程(Apoptosis pathway) ........................................ 43
附錄三:台灣牛樟芝 ............................................................. 44
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