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研究生:吳威翰
研究生(外文):Wei-Han Wu
論文名稱:牛樟芝子實體萃取物誘導肺癌細胞進行細胞凋亡之研究
論文名稱(外文):Extract from Taiwanofungus camphoratus fruiting bodies induce apoptosis in human lung carcinoma cell lines
指導教授:吳宗正吳宗正引用關係
指導教授(外文):Tzong-Zeng Wu
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:125
中文關鍵詞:凋亡肺癌
外文關鍵詞:apoptosislung
相關次數:
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有研究指出牛樟芝中的三萜類成分,可以誘導癌細胞產生凋亡現象,以抑制癌細胞的增生。但現今探討牛樟芝子實體成分對於肺癌細胞的影響,往往侷限於粗萃取物的測試。本研究嘗試以管柱層析分離牛樟芝子實體成分,經 HPLC 分析後, 得到 FK1~FK6 六種特徵區段的成分。經由細胞存活率測試,在 20μg/ml 樣品濃度時,無半抑制濃度 (IC50) 的樣品,但其 FK5 的抑制性較其他 5 種樣品高。經半製備 HPLC 分離 FK5 成分,得到 K5-1、K5-2、K5-3 三種成分。經細胞存活率測試,發現 K5-2 對於 A549 細胞之半抑制濃度 (IC50) 為 1.47μg/ml。在 12μg/ml K5-2 下,CCD-966SK 細胞存活率為 83% 以上 ( IC50 >12μg/ml ) 。顯示 K5-2 對癌細胞有高抑制性,對正常細胞較無毒殺性。在細胞週期、AnnexinV-FITC/PI 雙染、與TUNEL 實驗中,其流式細胞儀數據都顯示 K5-2促使 A549 細胞進行細胞凋亡。經由西方墨點法,証實K5-2之訊息傳遞路徑如下: K5-2 促使TRAIL ligand 表現增加,促進細胞膜上 DR4 蛋白表現增加。活化其下游路徑的 caspase 8,促進細胞中的 Bid 轉變成 tBid,tBid 及粒線體上的 Bax 表現增加、Bcl-2 蛋白表現下降,促使粒線體釋出 cytochrome C。Cytochrome C 釋出增加,會促進下游 caspase 3 活化。活化的 caspase3 會促使下游 caspase 7 蛋白活化,而活化的 caspase 7 會抑制 PARP 蛋白表現。PARP 蛋白無法正常運作,使得修復 DNA 的作用也會被抑制影響,導致產生 DNA 片段化現象產生。本研究初步証實主要透過細胞凋亡來抑制肺癌細胞A549的生長,未來應有機會發展成為治療肺癌藥物的潛力。
In some papers pointed out that the triterpenoids from Taiwanofungus camphoratus fruiting-bodies (TCFB) could induce apoptosis in cancer cells and inhibit cancer cells proliferation. However, previous studies of TCFB ingredients effect on lung cancer cells were often limited to crude extract of the test. This study attempted to isolate the TCFB entities by using column chromatography technique, the result showed six characteristic fractions ( FK1 ~ FK6 ) were obtained after HPLC analysis. Through the cell viability test, there were no half inhibitory concentration (IC50) of the above six fractions found at 20 μ g/ml concentration level, but the IC50 of FK5 higher than that of other 5 samples. Therefore further separated the FK5 fraction by semi-preparative HPLC, three features sections ( K5-1, K5-2, K5-3) were obtained. In cell viability test, the result showed the half inhibitory concentration (IC50) of K5-2was 1.47μg/ml for A549 cells. The CCD-966SK cell viability was at least 83% (IC50> 12μg/ml) in 12μg/ml of K5-2 concentration. K5-2 had high inhibitory for cancer cells but less cytotoxic to normal cells. All of the results of flow cytometric data ( cell cycle, AnnexinV-FITC/PI double staining , and TUNEL experiments ) showed that the K5-2 could induce A549 cell to apoptosis. The results of Western blot showed that the series of signal pathway of K5-2 was as followings: K5-2 induced TRAIL ligand expression and interacted with DR4 receptor induced caspase 8 activation. Caspase-8 activated tBid and then tBid stimulated Bax increased and decrease of Bcl-2 expression in mitochondria, induced cytochrome c from mitochondria release to cytoplasm. Cytochrome C release promoted the expression of downstream path of caspase 3. Activated caspase3 led to downstream caspase 7 activation, and caspase 7 inhibited PARP protein expression. PARP protein could not be normal operation, making the role of DNA repair were inhibited, lead to produce DNA fragmentation phenomenon. This study confirms K5-2 could efficiently inhibit the growth of lung cancer cell line A549 through apoptosis , it should have the opportunity to develop for treatment of lung cancer drug in the future.
中文摘要 Ⅰ
Abstract Ⅱ
目錄 Ⅳ
表目錄 Ⅴ
圖目錄 VI
第一章、 前言 1
第二章、 文獻回顧 3
第三章、 研究目的 25
第四章、 實驗材料與儀器 27
第五章、 實驗方法 35
第六章、 實驗結果 51
第七章、 結果與討論 63
第八章、 結論 71
第九章、 未來展望 73
第十章、 參考文獻 101
附錄 113
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