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研究生:蔡杰亨
研究生(外文):JIE-HENG
論文名稱:Rersveratrol甲基衍生物3,5,3'',4'',5''–pentamethoxystilbene (MR-5)抑制人類乳癌細胞MCF-7生長及細胞週期G1停滯之分子研究
論文名稱(外文):3,5,3,4,5’-pentamethoxystilbene (MR-5) , a Synthetically Methoxylated Analogue of Resveratrol, Inhibits Growth and Induces G1 Cell Cycle Arrest in Human Breast Carcinoma
指導教授:陳威仁陳威仁引用關係
指導教授(外文):Wei-Jen Chen
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生物醫學科學學系碩士班
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:70
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Resveratrol,是一種trihydroxy trans-stilbene,來自於葡萄及其他植物中,本身具有很強的抗氧化及抗腫瘤活性之天然化合物。3,5,3’,4’,5’–pentamethoxystilbene (MR-5)為一種以化學方式合成的resveratrol甲基衍生物,藉由官能基的修飾,是否和Resveratrol一樣,具有相同生物活性。因此,本研究之目的為探討白藜蘆醇及其甲基衍生物PTER, MR-3, MR-5,在人類乳癌細胞株MCF-7細胞中,對抑制細胞增生之效應及其可能之作用機轉作進一步探討。
研究結果顯示,在比較resveratrol和其PTER, MR-3, MR-5甲基衍生物實驗中,MR-5最能有效抑制人類乳癌細胞株MCF-7之細胞生長,降低細胞癌化程度,且呈現劑量及處理時間依賴性的效果,並造成細胞週期停滯於G1期。由西方墨點分析之結果顯示MR-5的生長抑制作用可能是與減少細胞內cyclin D1、cyclin D3、cyclin E和Cdk2、Cdk4和Cdk6蛋白量的表現以及增加p15、p16、p21和p27的表現量有關。由於上述分子在蛋白質表現量上的變化使得 Cdk2 和 Cdk4 激酶活性降低並導致RB無法被磷酸化。除此之外,MR-5也會影響人類乳癌細胞MCF-7內AKT、mitogen-activated protein kinase (ERK1/2)、p38 mitogen-activated protein kinase (p38 MAPK)和focal adhesion kinase (FAK)之活性,顯示MR-5抑制MCF-7增生與癌化的機轉,和這些訊息傳遞分子有關。
綜合上述, 本研究經由細胞模式結果顯示,MR-5活性具有抑制MCF-7乳癌增生之功效。本研究成果有助於了解resveratrol及其甲基衍生物對於抗乳癌活性上之保健功效,亦可提供未來開發結構相關新藥之重要參考。


3,5,3’,4’,5’-Pentamethoxystilbene (MR-5) is a synthetically met- hoxylated analogue of resveratrol and has been suggested to have
antitumor activity because of structural similarity to resveratrol. Herein, we investigate the antiproliferative effect of MR-5 in human breast cancer MCF-7 cells and demonstrate that MR-5 had a more potent inhibition on cell growth compared with resveratrol and other methoxylated derivatives. Exploring the growth-inhibitory mechanisms of MR-5, we found that it is accompanied by G1 cell cycle arrest, which coincides with a marked inhibition of G1 cell cycle regulatory proteins, including decreased cyclins (D1/D3/E) and cyclin dependent kinases (CDK2/4/6) and increased CDK inhibitors (CKIs) such as p15, p16, p21, and p27. Furthermore, the increase in CKI levels by MR-5 resulted in a concomitant increase in their interactions of CDK4 and CDK2, along with a strong inhibition in CDK4 kinase activity and the accumulation of hypophosphorylated Rb. MR-5 also modulated some critical kinase activities related to cell cycle regulation, including Akt, mitogen activated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), and focal adhesion kinase (FAK) in MCF-7 cells. In total, our results demonstrate that MR-5 affects multiple cellular targets that contribute to its antiproliferative activity in MCF-7 cells and provide novel information for synthetic chemists to design new antitumor agents with introduction of methoxylated group(s) in the basic compound.


目錄
壹、中文摘要………………………………………………1
貳、英文摘要………………………………………………3
參、縮寫表…………………………………………………4
肆、緒論……………………………………………………5
伍、文獻回顧………………………………………………7
陸、研究材料與方法………………………………………26
柒、實驗結果………………………………………………45
捌、討論……………………………………………………51
玖、附圖……………………………………………………55
拾、參考文獻………………………………………………64
圖次
圖一、Rersveratrol和Pterostilbene及其甲氧基衍生物化學結構圖..............................................55
圖二、R3、PTER、MR-3及MR-5抑制人類乳癌細胞株MCF-7的生長..............................................56
圖三、MR-5對人類周邊血管單核球細胞 (PBMCs)之細胞存活率及人
類乳癌細胞株MCF-7 LDH之釋放情形..............................................57
圖四、以固著非依賴性生長分析MR-5對人類乳癌細胞株MCF-7癌化的情形............................................58
圖五、Time-dependent和Dos-dependent 結果顯示MR-5誘使MCF-7細胞發生細胞週期 G1 時期停滯現象..............................................59
圖六、MR-5誘發MCF-7細胞G1 時期停滯的條件下,以西方墨點法觀察 G1 時期之相關蛋白表現情形..............................................60
圖七、MR-5誘發MCF-7細胞 G1 時期停滯的條件下,以西方墨點法及RT-PCR分析CDKIs相關蛋白和mRNA的表現量...........61
圖八、MR-5可抑制CDK2和CDK4複合體和CDK4激酶活性..............................................62
圖九、MR-5影響MCF-7細胞中AKT、ERK、FAK及P38磷酸化.63


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