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研究生:林佩姍
研究生(外文):Pei-Shan Lin
論文名稱:柳杉粗萃物及其生物活性化合物terpinen-4-ol 對肺癌之抗癌功能研究
論文名稱(外文):Anti-lung cancer effect of the crude extract and the bioactive compound terpinen-4-ol of Cryptomeria japonica on lung carcinoma cell lines
指導教授:林季千
指導教授(外文):Chi-Chen Lin
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:89
中文關鍵詞:4-&;#338204-&;#338204-&;#338204-&;#33820
外文關鍵詞:lung cancerCryptomeria japonicaterpinen-4-ol
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肺癌,高居全世界十大死因之首,是威脅人類健康的重要疾病。依據肺癌細胞生長的特性,可區分為小細胞癌和非小細胞癌。在台灣,非小細胞癌占肺癌患者中的88%。研究顯示柳杉樹皮分離之化合物能藉由抑制雄性激素接受體,而使得攝護腺癌細胞走向細胞凋亡。在本實驗中我們將探討柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol作用於肺癌細胞之分子機制。初步結果顯示柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol能誘導肺癌細胞進行細胞凋亡。肺癌細胞經過不同濃度之柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol作用後,在細胞生長試驗,肺癌細胞存活率隨著柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol濃度增加而遞減;由流式細胞儀細胞週期分析結果顯示,隨著柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol濃度增加,早期細胞凋亡和sub G1族群比例也隨之提高。而DNA片段化實驗更進一步證實,隨著柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol濃度增加,其細胞凋亡程度隨之增加。在分子機制方面,初步結果顯示柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol透過改變促凋亡蛋白和抗凋亡蛋白的比例,而活化粒線體相關的凋亡途徑,例如cytochrome c的釋放、caspase 9、caspase 3的活化而造成細胞凋亡。綜合以上實驗結果顯示柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol會藉由活化caspase-dependent pathway和調控MAPK pathway來達到抗癌的功能,期待柳杉葉部粗萃物及其生物活性化合物terpinen-4-ol能應用於未來治療癌症的研究方針上,帶給癌症患者一線生機。

Lung cancer is one of the most malignant cancer among the men and women. There are two main types of lung cancer: Small Cell Lung Cancers (SCLC) and Non-Small Cell Lung Cancers (NSCLC). Non-small cell lung cancers represent more than 80 percent of lung cancers in Taiwan. The report shows that a novel abietane diterpene, 6-hydroxy-5, 6-dehydrosugiol (HDHS), isolated from the stem bark of Cryptomeria japonica, was a potent AR antagonist in PCa cells. HDHS was found to be antiproliferative and proapoptotic against an ablation-resistant 22Rv1 tumor still expressing AR and responsive to androgen. This study was designed to exploit the molecular mechanisms of the extract of Cryptomeria japonica leaves and Cryptomeria japonica bioactive compound terpinen-4-ol induce apoptosis in lung cancer. From preliminary research and screening, the extract of Cryptomeria japonica leaves had inhibitory effect on lung cancer cells proliferation. The results indicated that the extract of Cryptomeria japonica leaves and terpinen-4-ol inhibited the cell viability of lung cancer cells by inducing apoptosis and increasing in the sub-G1 cell population. In addition, the extract of Cryptomeria japonica leaves and terpinen-4-ol also induced apoptosis in lung cancer cells, as determined by Annexin V staining assay. DNA fragmentation assay showed that the extract of Cryptomeria japonica leaves induced apoptosis. The extract of Cryptomeria japonica leaves and terpinen-4-ol increased the expression of pro-apoptosis Bax, but decreased the expression of anti-apoptosis Bcl-2 and subsequently triggered the mitochondrial apoptotic pathway (release cytochrome c and activation of caspase-9 and caspase-3). Taken together, these findings suggest that the crude extract of the leaves of Cryptomeria japonica and terpinen-4-ol can exhibit antitumor activity via caspase activation and MAPK regulation and can be effectively applied to cancer as a cancer chemopreventive agent.

誌謝 Ⅰ
中文摘要 Ⅱ
英文摘要 Ⅲ
目錄 Ⅴ
圖表目錄 Ⅷ
緒論 1
第一節 1
一. 肺癌 1
二. 肺癌之分類 1
三. 肺癌之治療方式 1
第二節 2
一. 柳杉 2
第三節 3
一. terpinen-4-ol 3
第四節 4
一. 細胞凋亡 4
二. 細胞凋亡之路徑 5
研究目的與動機 6
研究材料與方法 8
一. 細胞培養 8
二. 藥物處理 9
三. 藥物毒殺細胞存活率計算 10
四. 蛋白質萃取 10
五. 西方墨點法 12
六. 流式細胞儀分析Propidium Iodide染色 14
七. 細胞凋亡分析(Annexin V assay) 14
八. 細胞凋亡分析 (TUNEL assay) 15
九. DNA片段化分析(DNA Fragmentation assay) 15
十. 粒線體膜電位(Mitochondria membrane potential,△Ψm)之檢測 16
結果 18
第一節 18
一. 柳杉葉部粗萃物對非小細胞癌A549和老鼠肺癌LLC2之毒殺作用 18
二. 非小細胞癌A549和老鼠肺癌LLC2經柳杉葉部粗萃物處理後其形態上的變化 18
三. 柳杉葉部粗萃物誘導A549和LLC2細胞凋亡 18
四. A549細胞在柳杉葉部粗萃物的刺激下,走向intrinsic apoptosis pathway,引起細胞凋亡 20
五. A549細胞在柳杉葉部粗萃物的刺激下,走向p53-dependent pathway活化其下游基因,引起細胞凋亡 20
六. A549細胞在柳杉葉部粗萃物的刺激下,增加cleaved caspase-3、 cleaved caspase-9和cleaved caspase-PARP蛋白質表現 21
七.柳杉葉部粗萃物降低A549細胞內抗凋亡蛋白Bcl-2、XIAP、Survivin的表現量 21
八. 柳杉葉部粗萃物增加A549細胞內促凋亡蛋白Bax,Smac表現量 22
第二節 24
一. 柳杉葉部粗萃物hexane fraction對非小細胞癌A549和老鼠肺癌LLC2之毒殺作用 24
二. 非小細胞癌A549和老鼠肺癌LLC2經柳杉葉部粗萃物hexane fraction處理後其形態上的變化 24
三. 柳杉葉部粗萃物hexane fraction誘導A549和LLC2細胞凋亡 25
四. A549細胞在柳杉葉部粗萃物hexane fraction的刺激下,走向intrinsic apoptosis pathway,引起細胞凋亡 26
五. A549細胞在柳杉葉部粗萃物hexane fraction的刺激下,增加cleaved caspase-3、cleaved caspase-9和cleaved caspase-PARP蛋白質表現 26
六. 柳杉葉部粗萃物hexane fraction降低A549細胞內抗凋亡蛋白Bcl-2,XIAP,Survivin的表現量 27
七. 柳杉葉部粗萃物hexane fraction增加A549細胞內促凋亡蛋白Bax,Smac的表現量 27
第三節 29
一. terpinen-4-ol抑制腫瘤細胞和正常細胞生長 29
二. 非小細胞癌A549和CL1-0經terpinen-4-ol處理後其形態上的變化 29
三. terpinen-4-ol誘導非小細胞癌A549和CL1-0細胞凋亡 29
四. A549和CL1-0細胞在terpinen-4-ol的刺激下,減少Ras、Raf-1、p-ERK、ERK蛋白表現量 31
五. terpinen-4-ol 增加A549和CL1-0細胞內Cleaved-Caspase 9、Cleaved-Caspase 8、Cleaved-Caspase 3、Cleaved-PARP的表現量 31
六. terpinen-4-ol降低A549和CL1-0細胞內抗凋亡蛋白Bcl-2,XIAP,Survivin的表現量 32
七. terpinen-4-ol增加A549和CL1-0細胞內促凋亡蛋白Bax,Smac的表現量 32
八. terpinen-4-ol對A549和CL1-0細胞與細胞週期相關蛋白質的影響 33
討論 35
參考文獻 39
圖表 47

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4. 25、謝人俊,國際金融監理改革之新思維:總體審慎監理,存款保險資訊季刊第22卷4期,民國98年12月,頁123-193。
5. 25、謝人俊,國際金融監理改革之新思維:總體審慎監理,存款保險資訊季刊第22卷4期,民國98年12月,頁123-193。
6. 25、謝人俊,國際金融監理改革之新思維:總體審慎監理,存款保險資訊季刊第22卷4期,民國98年12月,頁123-193。
7. 7、李儀坤,美國金融改革與次級房貸危機之研討(下),信用合作第102期,民國98年10月,頁15-25。
8. 9、林盟翔,金融服務業橫向整合之困難與挑戰—金融與商業分離原則之定位與落實,中正財經法學雜誌創刊號,民國99年1月出版,頁77-122。
9. 7、李儀坤,美國金融改革與次級房貸危機之研討(下),信用合作第102期,民國98年10月,頁15-25。
10. 5、李桐豪,由功能性管制觀點探討我國金融體制的改革方向,存款保險資訊季刊第9卷2期,民國84年12月,頁23-36。
11. 6、李儀坤,避險基金之投資策略與金融監理,存款保險資訊季刊第20卷4期,民國96年12月,頁147-173。
12. 14、城仲模,我國已臻公司治理與金融監管的關鍵時刻,臺灣本土法學雜誌特刊,民國96年12月,頁2-8。
13. 14、城仲模,我國已臻公司治理與金融監管的關鍵時刻,臺灣本土法學雜誌特刊,民國96年12月,頁2-8。
14. 9、林盟翔,金融服務業橫向整合之困難與挑戰—金融與商業分離原則之定位與落實,中正財經法學雜誌創刊號,民國99年1月出版,頁77-122。
15. 24、劉一成,ABS及CDO商品盛行對金融監理之影響,存款保險資訊季刊第20卷4期,頁122-145。