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研究生:莊懿紅
研究生(外文):Yi-Hung
論文名稱:ERα調降PPARα的基因表現進而減緩Hep3B細胞增生的分子機制探討
論文名稱(外文):Estrogen receptorα (ERα) inhibits Hep3B cell proliferation mediates through the downregulation of peroxisome proliferator-activated receptorα (PPARα) gene expression.
指導教授:許立松
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
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:2007
畢業學年度:95
語文別:中文
論文頁數:91
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PPARα屬於細胞核內賀爾蒙受體的一員,在哺乳類動物中扮演著降低血脂肪,及平衡脂肪代謝的重要角色;但是,若長時間的在PPARα ligands(如類降血脂藥物Fenofibrate或脂肪酸類)刺激下過度活化PPARα,會導致肝癌的形成;肝癌(Hepatocellular carcinoma)是全世界五大癌症之一,據統計,全球每年有將近一百萬人死於肝癌,而台灣即屬於肝癌高發生及高死亡率的地區,由於肝癌的進程快速,且致病機轉為多重因素和多重步驟之過程,因此從分子生物醫學的角度尋找有利肝癌治療或診斷的機制,是重要且正積極進行的課題。而在實驗室先前研究曾發現,在Hep3B肝癌細胞中過量表現雌性素受體α(ERα),具有促進肝癌細胞凋亡的現象。另由文獻中得知,在MCF-7乳癌細胞中,PPARα的表現會被ERα所調控;所以在本實驗中,我們進一步探討,在人類Hep3B肝癌細胞株中,ERα是否可藉由調控PPARα的表現,達到降低肝癌的發生率,並進一步探討了ERα與PPARα之間相互的關係。Hep3B細胞處理了PPARα ligands (Fenofibrate),並採用RT-PCR及西方點墨法測量,同時發現p27蛋白表現量降低,cyclin D1及PCNA的蛋白表現量上升,且casp 3蛋白表現量也明顯的減少,其RNA亦有一致的表現情形;當進一步以MTT assay觀察,發現其細胞之增生存活率亦有明顯的增加;但在同時過量表現ERα時,則發現PPARα的表現量以及活性受到壓制,而原本被PPARα ligand所抑制的p27及casp 3之蛋白表現量則有明顯回升情形,而上升的cyclin D1及PCNA蛋白表現量則有明顯的降低;且再以MTT及TUNEL assay觀察時,發現當過量表現ERα時,PPARα所誘導的細胞增生情形受到壓制,而凋亡情形顯著上升;進一步以免疫沈澱法觀察,更發現ERα蛋白具有與PPARα結合的能力。綜合實驗的結果,得知,在人類Hep3B肝癌細胞株中,PPARα的表現除具有活化誘導Cell cycle進入S phase的能力,必具有降低Apoptosis的作用,進而導致細胞的增生;同時得知,ERα除了可調降PPARα的基因表現之外,還可能藉由蛋白與蛋白的交互結合作用影響了PPARα的活性,進而抑制Hep3B Cell cycle之進行並促進其細胞Apoptosis的發生。

Peroxisome proliferator-activated receptorα(PPARα) was a member of the nuclear receptor superfamily, has a central role in the regulation of numerous target genes encoding proteins that modulate fatty acid transport and catabolism. The lipid-lowering function of PPARα occurs across a number of mammalian species, thus demonstrated the essential role of this nuclear receptor in lipid homeostasis. In contrast, prolonged administration of PPARα ligands (Fenofibrate or fatty acid) causes hepatocarcinogenesis, specifically in rats and mice. Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world and is estimated to cause approximately half a million deaths annually. In addition, which has high incidence and fatality rates in Taiwan as well. Because of the rapid progress and multi-factor involvement of hepatocarcinoma, the improvements of diagnosis and the treatment outcome are still needed. Therefore, efforts have been made studying the biological mechanisms of hepatocarcinogenesis to these problems.
The previous studies in our lab have indicated that overexpressed estrogen receptor α(ERα) induced the apoptosis effets of Hep3B cells. Beside, it was reported that PPARα expression is regulated by ERα in MCF-7 cells. Therefore, we aim to determine the role of PPARα over-expression in hepatocarcinogenesis, and investigate how ERαregulate PPARα in Hep3B cells. Our data show that after treatment of fenofibrate, PPARα ligand decreases the protein levels of p27 and Caspase 3, and increase the protein levels of cyclin D1 and PCNA, results in the increase of cell proliferation observed by MTT assay. However, overexpressed ERα overcome the treatment of fenofibrate to increase the protein levels of p27 and Caspase 3, and decreases the protein levels of cyclin D1 and PCNA, results in the decrease of cell proliferation abserved by MTT assay and the increase of cell apoptosis measured by TUNEL assay. Moreover, ERα interacted directly with PPARα protein was observed by co-immunoprecipitation assay. Taken together, ERα might downregulate PPARα gene expression and protein activity via protein-protein interaction, to suppress cell proliferation and induce cell aopotosis of Hep3B cells.


誌 謝............I
目 錄............II
中文摘要..........1
Abstract..........3
壹、緒論..........5
貳、背景介紹......16
參、研究動機......27
肆、實驗方法......28
伍、實驗材料......42
陸、結果..........49
柒、討論..........59
捌、參考文獻......67
玖、圖表..........72
拾、附 錄........89



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