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標題: | 肝細胞中檳榔對於AP-1及NF-κB基因之調控 Mechanism of AP-1 and NF-κB activation and signaling by betel quid components in liver cells |
作者: | Ting-Ting Yang 楊婷婷 |
指導教授: | 鄭景暉 |
共同指導教授: | 吳慧琳(Hui-Lin Wu) |
關鍵字: | 檳榔子萃取物,AP-1,NF-κB,B型肝炎病毒X 蛋白,肝癌, Areca nut extract,AP-1,NF-κB,HBx,HCC, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | 嚼食檳榔在東南亞國家是一非常普遍之口腔嗜好,目前在全世界有六億之嚼食人口。嚼食檳榔已知為造成口腔白斑、口腔黏膜下纖維化、口腔癌及食道癌之主要危險因子之一。嚼食檳榔者由於可能會將唾液完全吞入上消化道,因此檳榔塊所產生的嚴重毒性作用也會在口腔以外的部位發生。許多的研究在動物實驗中發現檳榔子萃取物會對肝細胞造成基因毒性及細胞毒性。此外,轉錄因子AP-1及NF-κB與肝癌有很強烈的相關性,並在許多報告中皆指出肝炎病毒X 蛋白可活化AP-1 及NF-κB。近幾年來持續有針對於嚼食檳榔與肝癌相關性之流病報告,結果指出嚼食檳榔為一造成肝癌之獨立危險因子,並也對於慢性B 型肝炎病毒感染相關的肝癌有加成效果。由於台灣為B 肝病毒高帶原率的國家,因此本實驗欲針對檳榔成份合併B 型肝炎病毒兩大因素對於AP-1、NF-κB 轉錄因子分子調控機制與肝癌形成的關係進行探討。藉由報導基因分析我們發現檳榔子萃取物與檳榔素分別在800 μg/ml 及0.8 mM 的濃度下對於AP-1 及NF-κB 轉錄因子有活化之作用。在檳榔成份活化AP-1 的部分主要是經由Ras/Raf/ERK/MEK路徑,而在相同濃度的檳榔子萃取物作用之下,NF-κB 的部份則主要是經由PKC及MEK/ERK 但不經由Ras 路徑來達到活化。在B 肝病毒X 蛋白的存在下,對於檳榔子萃取物活化AP-1 及NF-κB 有協同活化效益,但對檳榔素則只有加成作用。我們利用RT-PCR 或西方墨點法研究檳榔子萃取物及檳榔素是否會誘導一些具有肝癌侵略性代表意義之相關指標分子的表現,結果發現檳榔子萃取物會增加tPA, PAI-1, IL-6 及IL-8 的基因表現,在HBx 存在下,IL-8 表現增加較為明顯; 檳榔素則會增加N-Cadherin, VCAM-1。總結來說,檳榔成份會對於轉錄因子AP-1及NF-κB 有活化作用,並在HBx 的存在下結果更加顯著,此外還會造成一些具有肝腫瘤侵略性之指標性分子之表現。因此我們提供了一個合併檳榔成份與肝炎病毒對於肝癌形成影響之可能分子機制,而對於臨床上之蘊含意義為在B 型肝炎病毒的帶原下,若加上嚼食檳榔之習慣可能會提高肝癌形成之危險性。 Betel quid (BQ) chewing is a common oral habit in Southeast Asian contries. There are about six hundred million BQ chewers in the world. The evidence of BQ chewing as one of the major risk factors leading to leukoplakia, oral submucous fibrosis, oral cancer and esophagus cancers have been well published. The BQ chewers may swallow saliva completely and therefore the severe toxic effects of BQ may occurs at sites other than the oral cavity. The animal studies have demonstrated that the betel quid components causes hepatotoxicity and the genotoxic effects of hepatocytes. In addition, transcriptional factors AP-1 and NF-κB has strong correlation with liver carcinogenesis and can be activated by X protein of the HBV. Recently, the epidemiology reports have shown that the habitual BQ chewing is an independent risk factors of hepatocellular carcinoma (HCC) and may have an addiditve effect with HBV infection. The HBsAg carrier rate in general population of Taiwan is one of the highest in the world. In our study, we like to investigate the AP-1 and NF-κB molecular mechanisms combine with HBV factors for the association of BQ components with the development of HCC. By using the luciferase assay, we found that the AN extract (ANE) 800 μg/ml and Arecoline (ACO) 0.8 mM induced AP-1 binding site activation mainly through Ras/Raf/MEK/ERK pathways. At the same ANE concentrations, NF-κB binding sites were activated mainly through PKC and MEK/ ERK pathways, but not the Ras pathways. In the presence of HBx protein, ANE-induced AP-1 and NF-κB is synergistically activated and with MEK/ERK pathways involvement. Compare with ANE, ACO-induced AP-1 and NF-κB activation in the presence of HBx protein only reach to the additive effects. At the mRNA level, some HCC invasiveness markers were induced by ANE (tPA, PAI-1 and IL-6 ) or ACO (N-Cadherin, VCAM-1). At the protein level, ANE alone or combine with HBx both induced IL-6 expression. However, the protein expression of IL-8 was only found in ANE or ACO stimulation in the presence of HBx. In conclusion, BQ components activate AP-1 and NF-κB transcriptional factors and induced the HCC invasiveness markers expression at the mRNA and protein level. ANE combine with HBV synergistically induced AP-1 and NF-κB activation. We provide the possible molecular mechanism of betel nuts chewing and combine with viral factors in the development of HCC. Our clinical implication is betel but chewing in HBsAg carriers may increase the risk of HCC development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26859 |
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