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研究生:陳馨瑩
研究生(外文):Sing-Ying Chen
論文名稱:Liriodenine抑制人類大腸癌細胞增殖的研究
論文名稱(外文):Liriodenine inhibits the proliferation of human colorectal carcinoma cells
指導教授:陳俊憲陳俊憲引用關係
指導教授(外文):Ching-Hsein Chen
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物醫藥科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
中文關鍵詞:Liriodenine細胞週期一氧化氮誘導型一氧化氮合成酶p53p21p27
外文關鍵詞:LiriodenineCell cycleNitric oxideiNOSp53p21p27
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Liriodenine,是萃取自蘭嶼烏心石(木蘭科)葉的生物鹼,是一種新發現的成分。研究發現由於具有DNA 崁入的能力,因此能抑制拓樸異構酶II (topoisomerase II)的活性。在活體試驗中,也發現能抗心律不整、抗菌、抗血小板活性等。先前研究指出Liriodenine能誘導肝癌細胞p53蛋白表現和NO產生,使細胞週期停滯在G0/G1期。
Liriodenine在抑癌的研究上對於大腸癌迄今尚無研究報告被發表,此研究將對Liriodenine造成人類大腸癌細胞抑制的作用機轉做探討,則這些資訊將可提供Liriodenine在抗大腸癌方面的應用。
利用流式細胞儀偵測發現Liriodenine 處理人類大腸癌細胞SW480細胞48小時後出現細胞週期G0/G1和G2/M期細胞週期停滯,並且使細胞內一氧化氮(NO)產生; 利用western blotting 方式偵測,發現Liriodenine處理後會增加SW480細胞iNOS的表現量,且影響細胞週期調控的相關蛋白: 使Cyclin D、Cyclin A,CDK表現量下降和CDK抑制劑p21、p27則表現量上升。用p53的抑制劑PFT-a抑制p53活性;用iNOS的抑制劑(S-ethylisothiourea; ETU)抑制NO的產生發現能阻止Liriodenine抑制細胞DNA合成的情形,而用ETU抑制NO的產生後,發現也能抑制P53表現,證明Liriodenine是經由NO產生及p53表現而造成抑制癌細胞增殖。利用siRNA抑制p53表現,發現p53、p27的表現也會受到抑制。利用siRNA抑制p21、p27表現,發現Liriodenine也會因p21、p27表現抑制而恢復癌細胞增殖,但不會抑制p53表現及NO產生。
綜合以上結果,Liriodenine會經由活化iNOS的表現使SW480細胞產生NO並活化p53、p21、p27表現進而調控細胞週期相關蛋白Cyclin D、Cyclin A,CDKs使細胞生長抑制造成G1和G2/M期拘留以及DNA合成抑制的情形。。
中文摘要…………………………………………………………………i
英文摘要……………………………………………………………i i i
目錄………………………………………………………………………v
圖表目錄………………………………………………………………i x
縮寫表………………………………………………………………x i i
第一章 文獻探討………………………………………………………1
第一節 前言………………………………………………………… 1
第二節 Liriodenine …………………………………………………1
第三節 細胞週期的調控(Cell cycle regulation)……………2
第四節 NO (nitric oxide)………………………………………9
第二章 研究目的…………………………………………………… 11
第三章 材料與方法………………………………………………… 13
第一節 材料 …………………………………………………… 13
第二節 方法………………………………………………………18
一、 細胞培養…………………………………………………18
二、利用流式細胞儀(Flow cytometry)分析細胞周期…… 18
三、利用流式細胞儀分析細胞內NO的表現………………… 19
四、利用西方墨點法分析細胞內蛋白表現………………… 19
五、利用RNAi技術抑制細胞內p53、p21、p27蛋白表現……23
六、統計方法………………………………………………… 25
第四章 結果………………………………………………………… 26
第一節 觀察Liriodenine對人類大腸癌細胞SW480細胞週期之影 響……………………………………………………………………… 26
第二節 觀察Liriodenine對人類大腸癌細胞SW480調控細胞週期相關 蛋白p53、p21、p27表現之影響………………………………………27
第三節 PFT-�悝磻螲53的活性對 Liriodenine抑制人類大腸癌細胞SW480細胞週期之影響…………………………………………………27
第四節 以p21 siRNA抑制p21的表現對 Liriodenine調控人類腸癌細胞SW480細胞週期的影響………………………………………………28
第五節 以p27 siRNA抑制p27的表現對 Liriodenine調控人類大腸癌細胞SW480細胞週期調控蛋白p53的作用…………………………… 29
第六節 ETU抑制NO的產生對 Liriodenine抑制人類大腸癌細胞SW480細胞週期之影響……………………………………………………… 29
第七節 以p53 siRNA抑制p53的表現對 Liriodenine調控人類大腸癌細胞SW480細胞週期調控蛋白p27的作用…………………………… 30
第八節 以p21 siRNA抑制p21的表現對 Liriodenine調控人類大腸癌細胞SW480細胞週期調控蛋白p53的作用…………………………… 31
第九節 以p27 siRNA抑制p27的表現對 Liriodenine調控人類大腸癌細胞SW480細胞週期的影響……………………………………………31
第十節 ETU抑制NO的產生對 Liriodenine誘導p53表現之影響……32
第十一節 觀察Liriodenine對人類大腸癌細胞SW480調控細胞週期相關蛋白cyclin A、cyclin E、cyclin D1表現之影響………………33
第十二節 觀察Liriodenine對人類大腸癌細胞SW480調控細胞週期相關蛋白CDK2、CDK4、CDK6表現之影響……………………………… 33
第十三節 觀察Liriodenine對人類大腸癌細胞SW480調控細胞週期相關蛋白磷酸化pRb表現之影響…………………………………………34
第十四節 觀察Liriodenine對人類大腸癌細胞SW480細胞iNOS表現之影響…………………………………………………………………… 34
第十五節 探討Liriodenine對人類大腸癌細胞SW480細胞內nitric oxide (NO)產生之影響……………………………………………… 35
第十六節 探討ETU抑制Liriodenine對人類大腸癌細胞SW480細胞內nitric oxide(NO)產生之影響……………………………………… 36
第十七節 以p21和p27 siRNA抑制p21和p27的表現對 Liriodenine調控人類大腸癌細胞SW480細胞內nitric oxide(NO)產生之影響……………………………………………………………………… 37
第四章 討論………………………………………………………… 38
第五章 參考文獻…………………………………………………… 45
第六章 圖…………………………………………………………… 55
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