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研究生:許文建
研究生(外文):Wen-Chien Hsu
論文名稱:Menadione誘發人類乳癌細胞MDA-MB231細胞週期G0/G1停滯之分子機制研究
論文名稱(外文):Studies on the molecular mechanisms of Menadione-induced G0/G1 cell-cycle arrest in MDA-MB 231 Human Breast Cancer cells
指導教授:何元順
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗生物技術學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:76
中文關鍵詞:維生素K3人類乳癌細胞
外文關鍵詞:MenadioneMDA-MB 231
相關次數:
  • 被引用被引用:1
  • 點閱點閱:233
  • 評分評分:
  • 下載下載:66
  • 收藏至我的研究室書目清單書目收藏:0
Vitamin K是脂溶性維生素,以結構的不同分成K1(phylloquinone)、K2(menaquinones)、K3 (menadione),其中K1來自日常飲食中的綠葉蔬菜,K2來自腸道中之細菌合成,而K3則是合成維生素,維生素主要功能為催化肝臟合成凝血因子prothrombin促進凝血機制。由過去文獻報告指出,Vitamin K也具有抗癌的效果,但作用機制不是完全清楚,目前了解依作用機制分成2種模式,一則是氧化模式(oxidative model),透過氧化還原的方式大量產生過氧化物(ROS),增加細胞氧化的負擔而進一步造成細胞死亡,另一則是非氧化模式( non oxidative model)由從轉錄因子來探討,Vitamin K會促進特定的轉錄因子表現進而調控細胞週期停滯及細胞凋亡。實驗中利用低濃度(5.8μM ) menadione對人類乳腺上皮細胞癌是否具有抗癌的效果以及分子作用機制討論。MTT及生長曲線的實驗,發現menadione在低濃度且長時間作用情況下,對在於人類乳腺上皮細胞癌(MDA-MB 231)是有明顯的抑制作用,而在正常乳腺細胞(MCF-10A)則沒有影響,在分子轉錄上及蛋白質表現方面也發現低濃度menadione處理24時能使MDA-MB 231誘發p21基因及蛋白表現量增加及cyclin E、CDK2抑制,再經由流式細胞儀分析也發現低濃度 menadione 處理24小時能使MDA-MB 231細胞週期GO/G1比例上升,造成細胞週期停滯,進而抑制細胞生長。而在調控p21基因表現的調節者部分,雖然沒有進一步發現,但也讓我們在未來可以朝此方向將menadione誘發p21基因表現的途徑呈現更完整。
Vitamin K is a family of structurally similar fat-soluble
2-methyl—1,4-naphtho-quinones,including phylloquinone(K1),
menaquinones(K2),and menadione(K3). K1 is found in many green leafy vegetables. K2 is produced by a vast array of intestinal bacteria in human body. Menadione (K3) is not considered a natural vitamin K ,but rather a synthetic analoque acts as a provitamin .Vitamin K acts as a cofactor in normal blood coagulation from the post translation modification of a number of plasma proteins such as prothrombin. Vitamin K has been the focus of considerable research demonstrating an anticancer potential.However, yet the mechanisms of action remain unclear. A number mechanisms has been proposed and focused on the oxidative model which increased redox-cycling of menadione and the production of ROS surpasses the oxidative capacity of the cell, resulting in cell death. On the other hand, the non- oxidative model focuses on the modulation of transcriptionl factors which in turn induce cell cycle arrest and apoptosis. In our study, we found menadione has anticancer potential on human breast adenocarcinoma MDA MB 231 by weather cell cycle arrest, anti proliferation, or apoptosis. Low concentration of menadione (5.8μM) significant inhibited growth curve on breast cancer cells (MDA MB 231) but not breast cells (MCF 10A) by performing MTT and trypan blue cell counting. RT-PCR and western blotting showed menadione increase p21 gene and protein expressions but decrease cyclin E and CDK2 protein expression resulting in cell cycle G0/G1 arrest by flow cytometry. In the future, next step will be find out the key molecular proteins that regulate p21 expression and complete the whole view of mechanism by low concentration of menadione .
目錄-1
圖目錄-3
中文摘要-4
英文摘要-6
第一章 緒論-8
第二章 文獻探討-11
第一節 前言-12
第二節 細胞週期-12
第三節 細胞週期停滯p53/p21-15
第四節 維生素K/ K3(Menadione))-17
第五節 轉錄因子Sp1-20
第三章 研究方法-22
第一節 實驗材料-23
第二節 實驗方法-29
(1) Cell culture (細胞培養及藥物處理)-29
(2) MTT cell viability assay (細胞存活率分析)-29
(3) Cell growth curve (細胞生長曲線)-30
(4) RT-PCR (反轉錄聚合連鎖反應)-30
(5) Flow cytometry (流式細胞儀分析細胞週期)-31
(6) Western blotting assay (西方墨點法)-33
(7) Nuclear and cytoplasmic extraction (核質分離)-34
(8) Chromatin Immunoprecipitation assay (染色質免疫沉澱法)
(9) Polymerase Chain Reaction(聚合連鎖反應)-36
(10) 分析方法-36
第四章 分析結果-37
第一節 Menadione 對MDA-MB 231 及MCF-10A細胞生長的影響-38
第二節 Menadione誘發MDA-MB 231細胞p21基因表現-39
第三節 建立MDA-MB 231細胞週期及Menadione誘發G0/G1細胞靜止-39
第四節 Menadione誘發MDA-MB 231細胞週期停滯其細胞週期蛋白的變化-40
第五節 調控p21基因的上游調節者-核質分離表現情形-42
第六節 調控p21基因的上游調節者-染色質免疫沈澱表現情形
第五章 討論-44
圖表-50
參考文獻-69
附錄-75
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