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研究生:王學孝
研究生(外文):Hsueh-Hsiao Wang
論文名稱:動脈粥狀硬化過程中保護因子之研究I.β2-glycoproteinI基因上游5'端調控區之選殖與功能鑑定,II.探討多元不飽和枝防酸及中草藥活化過氧化酶體增生活化接受器之作用機制王學孝著
論文名稱(外文):Studies on the protective factors in the process of atherosclerosis
指導教授:姜安娜姜安娜引用關係
指導教授(外文):An-Na Chiang
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
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:2004
畢業學年度:92
語文別:中文
中文關鍵詞:動脈粥狀硬化脂蛋白元肝臟轉錄因子基因調控多元不飽和脂肪酸啟動子
外文關鍵詞:atherosclerosisapolipoproteinhepatic nuclear factorgene regulationpolyunsaturated fatty acidpromoter
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動脈粥狀硬化這個最重要的心臟血管疾病也是長久以來已發展國家中主要的致死原因,為了了解如何減緩動脈粥狀硬化之發生,本研究主要在探討抗動脈粥狀硬化作用機制之內生性和外源性的因子。
β2-glycoprotein I (β2-GPI)是主要由肝臟合成的血漿醣蛋白,但現今β2-GPI基因層次的調控仍未闡明,為了瞭解β2-GPI基因之調控機制,我選殖並分析了 4.1 kb 長度的β2-GPI基因5''端調控區,並利用刪除及定點突變法發現了一段非標準的TATA box及HNF-1結合區域主導β2-GPI近位啟動子的活性,電泳位移實驗確認了HNF-1α這個蛋白質與在TATA box下游HNF-1序列間的交互作用,又進一步在肝癌細胞Huh7與不表現HNF-1的HeLa細胞株中外送HNF-1α表現質體證明了HNF-1α橫貫活化β2-GPI啟動子的能力,這些結果顯示非標準的TATA box 及HNF-1結合序列在細胞表現特異性上的重要角色;此外我們也分析了氧化壓力對β2-GPI基因表現的影響,在肝癌細胞中過氧化氫及phorbol ester會降低β2-GPI的啟動子活性並改變細胞核蛋白質與β2-GPI近位啟動子間的作用模式,但在心臟動脈血管平滑肌細胞中,氧化壓力卻會增加β2-GPI的表現。這顯示在氧化壓力下β2-GPI會呈現一差異性的表現模式,更指出了在有代謝性症狀與疾病患者體內β2-GPI之表現變化或有臨床上的重要性。
動脈粥狀硬化之病理過程會受魚油或中草藥等食物因子的影響,最近的研究顯示飲食中的多元不飽和脂肪酸(PUFAs)會調控過氧化酶體增生活化接受器(PPARs)的活性,PPARs是細胞核內受配體活化之轉錄因子,它會與視黃酸接受器(RXR)形成異二聚體後與PPAR調控區域(PPREs)結合影響其標地基因之表現。由於PPARs生理功能上所調控的基因被認為具有抗動脈硬化的潛力,我們又在apoE剔除小鼠中發現魚油能降低動脈粥狀硬化損傷,因而進一步分析PUFAs對脂蛋白元AI (apoAI)基因上游受PPARα調控之序列(PPREα)及帶有肝臟X接受器α (LXRα)基因上游受PPARγ調控之序列(PPREγ)之橫貫活化能力。利用具有基因啟動子調控區及luciferase之基因載體篩選PPARα及PPARγ之活化配體,結果顯示n-3 PUFAs能透過PPREγ活化LXRα基因表現以及增加細胞中ATP binding cassette A1 (ABCA1)的表現,這可能降低泡沫細胞中膽固醇的堆積。此外亦在中草藥之水及酒精萃取物中發現具有活化apoAI及LXRα標的基因之成分,此種報導質體分析系統具有應用食療保健在防治動脈粥狀硬化研究之潛力。
Atherosclerosis, the most clinical manifestation of coronary artery disease, is responsible for the main cause of death in developed countries. To understand the protective interventions, we focus on endogenous and exogenous factors contributing to anti-atherogenesis.
β2-Glycoprotein I (β2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The regulation of β2-GPI gene expression has not been clarified. To gain more insight into the control mechanism of β2-GPI gene expression, we cloned the 4.1-kb 5′-flanking region and characterized the proximal promoter of the β2-GPI gene. Truncation and site-directed mutageneses of putative cis-elements within this region showing an atypical TATA box and a hepatic nuclear factor-1 (HNF-1) element were both essential for the β2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1α with the HNF-1 site residing downstream of the TATA box. Cotransfection of β2-GPI promoter-luciferase vector with HNF-1α expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the trans-activation effect of HNF-1α on β2-GPI promoter activity. These data suggest that the atypical TATA box and HNF-1 cis-element are both critical for β2-GPI transcription and the HNF-1α may play an important role in cell-specific regulation of β2-GPI gene expression. Moreover, we investigated the role of oxidative stress in the regulation of β2-GPI expression. In hepatoma cells, hydrogen peroxide and phorbol ester decreased the transcriptional activity of β2-GPI reporter constructs and altered the binding pattern between nuclear proteins and human β2-GPI proximal promoter. However, the mRNA levels of endogenous β2-GPI were activated by phorbol ester and hydrogen peroxide treatment in human coronary artery smooth muscle cell. The differential regulation of β2-GPI under oxidative stress, implying that the interindividual variation of β2-GPI levels in subjects with various metabolic syndrome and disease states may have clinical importance.
The process of atherosclerosis can also be modulated through dietary factors. Recent studies have shown that polyunsaturated fatty acids (PUFAs) regulate the activity of peroxisome proliferator-activated receptors (PPARs). PPARs are a family of ligand-activated nuclear transcriptional factors that bind to its peroxisome proliferator response elements (PPREs) as a heterodimer with a retinoid X receptor (RXR) and regulate their target gene expression. From recent reports, the physiological function of PPAR-mediated gene expression might have anti-atherosclerosis potential. We used apoE knockout mice to assess the role of fish oil in the development of atherosclerosis and the result showed that supplementation with fish oil reduced atherosclerotic lesions. We further investigated whether PUFAs contribute to the trans-activation of apoAI and LXRα genes via PPREα and PPREγ, respectively. Transient transfection experiments with plasmids containing PPREs in front of the luciferase reporter gene is responsible for the screening PPARα and PPARγ agonists. The results showed that n-3 PUFAs transactivated PPREγ of LXRα and enhanced ATP binding cassette A1 (ABCA1) expression, which might decrease cholesterol accumulation during foam cell formation. Moreover, water and alcohol extract of several herbal medicines has implicated in the trans-activation of apoAI and LXRα target genes. This reporter assay system creates a potentially opportunity for the dietary manipulation of atherosclerosis.
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