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標題: | 鑑定受幽門螺旋桿菌感染之胃腺癌上皮細胞內的激酶Akt受質 Identification of Akt Substrates Involved in Helicobacter pylori Infected Gastric Adenocarcinoma Epithelial Cells |
作者: | Mei-Chun Lin 林玫君 |
指導教授: | 周綠蘋(Lu-Ping Chow) |
關鍵字: | 胃癌,幽門螺旋桿菌,激酶,Akt,免疫共沈澱,蛋白質體學, Gastric cancer,Helicobacter pylori,Akt,Co-IP,Proteomics, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 胃癌是其中一種屬於高發生率及高死亡率的癌症類型,罹患胃癌的影響因子包含生活習慣、飲食習慣或是曾有胃部疾病史等。除此之外,在西元1994年被認定屬於一級致癌因子的幽門螺旋桿菌 (Helicobacter pylori) 也是與胃癌發生相關的病原體,根據先前的研究指出,感染幽門螺旋桿菌與胃癌發生之間具有高度的相關性。幽門螺旋桿菌其外形呈現螺旋狀,屬於革蘭氏陰性菌。在西元1982年,Barry J. Marshall及Robin J. Warren成功從罹患胃炎及消化性潰瘍的病人胃部檢體分離出幽門螺旋桿菌,並且確立該細菌為誘發胃炎及消化性潰瘍發生的重要因子,然而幽門螺旋桿菌感染所引發的宿主反應及細胞內訊息傳遞機制仍不清楚。
Akt屬於Serine/Threonine激酶,會去磷酸化各種參與在細胞週期、細胞存活及細胞生長等相關的蛋白質。PI3K/AKT路徑的活化很常發生在人類惡性腫瘤內,其中胃癌也有同樣的現象,然而對於其切確的功能仍不清楚。現今認為激酶Akt對於腫瘤生成的過程扮演重要的角色。再者先前的研究發現在幽門螺旋桿菌感染的情況下,會促使PI3K/AKT路徑的活化。因此我們希望可藉由質譜蛋白質體學 (mass spectrometry-based proteomic approach) 的方法鑑定幽門螺旋桿菌感染時,激酶Akt會去磷酸化的下游受質有哪些,並探討這些分子對於胃癌致癌機轉的重要性。 為了鑑定激酶Akt的受質,我們使用液相層析串聯質譜儀 (liquid chromatography tandem mass spectrometry) 鑑定利用免疫共沈澱 (co-immunoprecipitation) 技術分離出來的激酶Akt複合物。藉由比較未感染幽門螺旋桿菌及有感染幽門螺旋桿菌激酶Akt免疫共沈澱複合物之間的差異性,我們鑑定到37個激酶Akt可能的交互作用個體。接著我們使用生物資訊統計軟體分析鑑定到的交互作用個體並以西方點墨法 (western blotting) 進行驗證。我們發現幽門螺旋桿菌感染時,EphA2在Ser 897位置的磷酸化有增加的現象。此外,當抑制EphA2蛋白質表現量時,會減弱細胞移動的能力。 未來我們將會對其他鑑定到的蛋白質磷酸化程度進行驗證並且探討其生理作用,期望可以對PI3K/AKT路徑參與在幽門螺旋桿菌感染因而造成胃癌的致癌機轉有更深入的瞭解。 Gastric cancer, one of the high incidence rates and mortality types of cancer, results from lifestyle habits, dietary factors, or a history of disorders of stomach. Besides these factors, Helicobacter pylori, classified as a class Ⅰ carcinogenic agent in 1994, is also an important pathogen related to gastric cancer. According to previous studies, the infection of Helicobacter pylori has a high correlation with the incidence of gastric cancer. Helicobacter pylori belongs to gram-negative bacterium having spiral shape. In 1982, Barry J. Marshall and Robin J. Warren isolated Helicobacter pylori from the stomach specimens of gastritis and peptic ulceration patients, and established that the infection of Helicobacter pylori is an important factor inducing the occurrence of gastritis and peptic ulceration. However, the host responses and regulation of signaling transduction during Helicobacter pylori infection are still not understood. Akt, as a serine/threonine kinase, phosphorylates multiple substrates involved in cell cycle progression, cell survival, cell growth and so on. Activation of PI3K/AKT pathway is one of the most common molecular events in human malignancies including gastric cancer, although the precise function remains unclear. It seems that Akt may play an emerging role in tumorigenesis. Moreover, it has been reported that PI3K/AKT pathway is also activated during Helicobacter pylori infection. Therefore, we aim to identify the downstream targets of Akt that may be phosphorylated during Helicobacter pylori infection by proteomics approach to investigate the carcinogenesis of gastric cancer. In order to identify Akt substrates, we analyzed Akt co-IP complex by LC-MS/MS (liquid chromatography tandem mass spectrometry). By comparing the differences of Akt co-IP complex between AGS cells and AGS cells infected with Helicobacter pylori, we identified 37 Akt interacting proteins. Then we analyzed these potential interacting proteins with bioinformatics software and validated the results with western blotting. Furthermore, among these proteins, we found that EphA2 Ser 897 phosphorylation was regulated in response to Helicobacter pylori infection. In addition, after knocking down EphA2, the ability of cell migration was decreased. In the future, we will further validate the phosphorylation level of other identified possible Akt substrates and their physiological roles, hoping to demonstrate the involvement of PI3K/AKT pathway in gastric carcinogenesis induced by Helicobacter pylori. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/5933 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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