中文摘要

胃幽門螺旋桿菌是一種微厭氧的革蘭氏陰性菌，一般寄生在人體的胃壁。全球幾乎有一半的人口感染胃幽門螺旋桿菌，但是目前對於胃幽門螺旋桿菌的致病機制仍然不很清楚。根據以往的研究，許多細胞內的訊息傳遞是藉由改變細胞內蛋白質的磷酸化來達成調控的目的；因此推測胃幽門螺旋桿菌的侵入也許會造成寄主細胞內某些蛋白質磷酸化的程度增加或減少。為了驗證此一假設，我使用可偵測蛋白質酪胺酸處是否有磷酸化修飾的抗體，以比較有胃幽門螺旋桿菌感染的人類胃上皮細胞內蛋白質與未受感染的人類胃上皮細胞內蛋白質有何差異。結果顯示當人類胃上皮細胞被胃幽門螺旋桿菌感染之後，細胞內某些蛋白質酪胺酸處的確有磷酸化增加或減少的情形，其中的一些甚至可能是胃幽門螺旋桿菌的細胞毒性相關基因A (cagA)所產生的蛋白造成的磷酸化改變。為了進一步鑑定這些蛋白質，我使用可純化蛋白質酪胺酸處磷酸化修飾的抗體，以免疫沉澱法將它們純化出後解析在膠上並以SYPRO Ruby染色法分析。最後，以質譜儀分析了選出的八個可能有磷酸化改變的蛋白質條帶，其中的M2型丙酮酸激酶 (M2-type pyruvate kinase)是最有可能在胃幽門螺旋桿菌感染後酪胺酸處磷酸化增加的蛋白質。

Abstract

Helicobacter pylori, a spiral, gram-negative, microaerophilic bacterium that colonizes the human stomach, is thought to infect about half of the world’s population. However, little is known about the molecular mechanisms by which H. pylori exerts the pathogenesis. Base on our knowledge, most of the proteins involved in the signaling events might be regulated by phosphorylation. To determine whether cellular proteins are phosphorylated after H. pylori infection, the proteins being tyrosine-phosphorylated in AGS cells after H. pylori infection were examined by Western blot analysis. I found that several proteins in AGS cells were phosphorylated or dephosphorylated at tyrosine residues after H. pylori infection. Many of these tyrosine-dephosphorylated proteins belong to CagA (cytotoxin-associated gene A)-dependent event. To further identify these tyrosine-phosphorylated or dephosphorylated proteins, I applied the immunoprecipitation technique to isolate these proteins and resolved them by SDS-PAGE followed by SYPRO Ruby staining. Eight bands of interest were excised from the gel and subjected to in-gel tryptic digestion. After analyzing by MALDI-MS, M2-type pyruvate kinase was identified as the most possible protein which was phosphorylated in response to H. pylori infection.

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