臺灣博碩士論文加值系統

咖啡酸苯乙酯(caffeic acid phenethyl ester, CAPE) 這類化合物具抑制癌細胞生長，抗氧化活性，抗病毒、促進免疫活性等性質。由於CAPE具有這些性質，其相關衍生物亦因而成為具有發展潛力之化合物及研究題材。 關於這類化合物之研究雖然很多但是其詳細的作用機轉仍有待釐清。我們研究發現CAPE在細胞內清除過氧化氫導致胞內氧化還原平衡破壞、降低細胞內Mcl-1蛋白含量、活化caspase-8 導致細胞凋亡。由於實驗發現CAPE可導致細胞週期停止在S-phase，所以在本研究中，我們進一步分析CAPE導致S-phase停滯之作用機制。由於E2F-1過量表現與S-phase停滯可能有關。因此，我們也探討E2F-1在CAPE作用所扮演之角色，我們發現CAPE可導致E2F-1，p21，Apaf-1之表現量增加，為了確認E2F-1，Apaf-1及p21蛋白之表現量增加及Mcl-1表現量下降，都是與E2F-1有關，我們將Mcl-1，E2F-1及Apaf-1之基因啟動區(promoter)植入螢光酶報告載體進行分析，以瞭解CAPE是否確實可以經由增加E2F-1蛋白質之表現及與E2F-1序列結合而影響Mcl-1、Apaf-1等基因之表現。結果顯示，CAPE確實能經由提高E2F-1表現而影響下游基因之表現而產生各種作用。此外以RNAi方法來抑制細胞內E2F-1因子之表現來，也證明CAPE抑制NF-kB之作用與細胞內E2F-1表現量呈現相反關係。綜合上所述，本研究顯示CAPE對細胞生長及凋零之作用與其影響E2F-1因子表現有密切關係。

Proplis, a honeybee-hive products, exhibits anti-inflammatory, antiviral, immunostimulatory and carcinostatic activities. Known for the variety of its beneficial effects, it has been a popular folk medicine through the ages. The active ingredient of propolis was identified as caffeic acid phenylether ester ( CAPE). CAPE was shown to involved in many cellular processes to induce differential toxicity to cancer cells versus normal cell lines. Despite the knows properties of these compounds in modulation of cellular processes, the molecular basis of their action remains to be clarified. Previous studies in our lab indicated that CAPE-mediated its action through lowering intracellular hydrogen peroxide levels, activation of caspase 8 and decrease Mcl-1 protein level. To investigate the action mechanism of CAPE, we analyzed the effect if this compound on cell cycle progression and found that the CAPE arrested human cervical ME180 cells at S-phase. In addition, the S-phase arrest is associated with increased expression of E2F1, p21 and Apaf-1 proteins and decreased expression of Mcl-1 protein. To verify the role of E2F1 in CAPE-mediated cell growth arrest, we constructed E2F1, Apaf-1 and Mcl-1 promoter-luciferase reporters and analyzed by transient transfection studies. Ours results indicated that the presence of E2F1 binding sequence (EBS) in the reporters is essential for alteration of promoter activity in CAPE-treated cells. Using electrophoretic mobility shift assay (EMSA), we showed that the exposures of cells to CAPE resulted in enhanced E2F1 binding to the wild type EBS but not to the mutated EBS oligos. Further studies using RNAi techniques to suppress endogenous expression with the inhibitory effect of CAPE on the transcriptional activity of E2F1 overexperssion with the inhibitory effect of CAPE on the transcriptional activity of NF-kB factor. Taken together, our study presents evidence that indicated the crucial role of E2F1 protein factor in the cellular actions of CAPE. We hope that our study would help to elucidate the molecular basis and therapeutic potential of this agent.

目 錄……………………………………………… I
圖表目錄……………………………………………… II
縮 寫 表……………………………………………… III
中文摘要……………………………………………… IV
英文摘要……………………………………………… V
緒 論……………………………………………… 1
材料與方法…………………………………………… 9
結 果……………………………………………… 33
討 論……………………………………………… 40
結 論……………………………………………… 46
參考文獻……………………………………………… 47
圖 表……………………………………………… 52

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