中文摘要
戴奧辛是存在於環境中的有機污染物，而且對於生物體具有多種不同的毒性。流行病學調查中可以知道，暴露到戴奧辛會增加得到慢性阻塞性肺病、肺功能降低、肺癌的危險性。目前已經知道戴奧辛中毒性最強的TCDD是一種會和戴奧辛受器(AhR)結合的ligand-activated轉錄因子，其可能改變基因的表現。而且我們已經證實在人類肺腺癌中，戴奧辛受器(AhR)會過量表現。因此需更進一步地瞭解戴奧辛受器(AhR)在肺腺癌中所扮演的角色。近年來，蛋白質體學發展出許多新技術，可用來分析混合物蛋白質的組成。因此為了要研究人類肺腺癌細胞(H1355)受戴奧辛受器(AhR)表現影響的蛋白質體，我們將戴奧辛(TCDD)加入受到RNA干擾AhR基因而沈默不表現的人類肺腺癌細胞(H1355)中。利用二維電泳的方法分析人類肺腺癌細胞(H1355)蛋白質萃取物，經過銀染後可以看到有超過500個蛋白質的點出現在膠上。經過比較受到RNA干擾與不受干擾的二維電泳圖譜之後，發現有差異蛋白質有55個，其中有27個為up-regulated，28個為down-regulated。有17個蛋白質經由液相層析串聯式質譜儀被鑑定確認，這些被鑑定的蛋白質中包括chaperones、結構蛋白、訊息傳遞蛋白、骨架蛋白、代謝性蛋白質、以及ubiquitin proteasome system protein。上述這些蛋白質中，利用西方點墨法確認了Galectin-1、HSP27、beta-actin 和GRB10，我們發現在受到AhR RNAi干擾的細胞中，Galectin-1、HSP27、beta-actin這些蛋白質表現上升，然而GRB10表現下降。給予戴奧辛(TCDD)會減少GRB10的蛋白質表現。這些結果證實了蛋白質體技術運用在體外鑑定蛋白質是有效的。它同時也顯示在肺細胞中戴奧辛受器(AhR)的表現會影響某些蛋白質之表現。未來我們將會研究這些被改變的蛋白質在肺腺癌中所扮演的功能。

Abstract

Dioxins are persistent organic pollutants in the environment and have diverse toxic effects in living organisms. Epidemiological studies suggested that exposure to dioxins highly enhanced the risk of pulmonary diseases, including lung cancer. It is well known that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent dioxin, binds to aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which subsequently alters gene expression. Furthermore, it has been demonstrated that AhR over-expressed in human lung adenocarcinoma. Therefore, the role of AhR in lung adenocarcinoma remained to be clarified. Recently, many proteomic technologies are developed to analyze proteins mixtures. To investigate the proteome of human lung adenocarcinoma cells H1355 affected by AhR expression, we used RNA interference (RNAi) technique to silence AhR gene in H1355 cells followed by TCDD treatment. Proteins extracted from H1355 cells were displayed by two-dimensional gel electrophoresis (2DE). More than 500 protein spots were visualized after silver staining. The comparison between 2DE maps with RNA interference and without RNA interference showed alterations of 55 spots, of which 27 were up-regulated and 28 were down-regulated. Seventeen proteins were identified with LC-MS/MS. The identified proteins include chaperones, structural proteins, signal transduction proteins, cytoskeleton proteins, metabolism proteins and ubiquitin proteasome system protein. Among these proteins, Galectin-1, HSP27, beta-actin and GRB10 were confirmed with Western immunoblot. We found that Galectin-1, HSP27 and beta-actin were up-regulated, but GRB10 down-regulated, in AhR-interfered cells. TCDD treatment further reduced GRB10 protein levels. These results demonstrated that proteomic techniques are useful in identifying proteins in vitro. It also showed that AhR expression influenced certain protein levels in lung cells. We will further investigate the function of these altered proteins in lung adenocarcinomas.

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