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Title: | 分析幽門螺旋桿菌感染所誘發磷酸化蛋白質於細胞位置之改變 Subcellular Localization Change of Phosphoproteins Induced by Helicobacter pylori infection |
Authors: | Shu-Han Yu 游舒涵 |
Advisor: | 周綠蘋(Lu-Ping Chow) |
Keyword: | 幽門螺旋桿菌,胃癌,磷酸化蛋白質體學,共軛焦顯微鏡,Akt,β-catenin, Helicobacter pylori,gastric cancer,phosphoproteome,confocal,Akt,β-catenin, |
Publication Year : | 2009 |
Degree: | 碩士 |
Abstract: | 當幽門螺旋桿菌感染宿主細胞時,宿主細胞會產生細胞形態改變、細胞增生、發炎或是抗細胞凋亡等反應。近年來有許多科學家均致力於研究細胞感染幽門螺旋桿菌後訊息傳遞路徑的調控,試圖解釋幽門螺旋桿菌與消化道疾病的顯著關連性。由於蛋白質磷酸化快速且可逆的調控特性,使得細胞能夠更即時地反應刺激並傳遞訊息,因此本實驗室利用磷酸化蛋白質體學技術研究胃腺癌上皮細胞被幽門螺旋桿菌感染時細胞內所誘發的蛋白質磷酸化變異,先利用immobilized metal affinity chromatography (IMAC) 純化細胞質中的磷酸化蛋白,並將這些受幽門螺旋桿菌感染後磷酸化的蛋白質以ESI-LC/MS/MS進行鑑定再以訊息傳遞資料庫Ingenuity Pathways Analysis (IPA)進行分析,推測出可能的訊息傳遞網路。
IPA軟體預測出當幽門螺旋桿菌感染後會促使Glycogen Synthase Kinase 3β (GSK3β)磷酸化。西方墨點染色實驗結果指出,當細胞感染幽門螺旋桿菌後確實會透過磷酸化來調控Akt, GSK3β, Cyclin D1等訊息傳遞路徑。本實驗進一步利用共軛焦顯微鏡來觀察這些被幽門螺旋桿菌誘發磷酸化蛋白是否產生分布位置的改變,結果發現當幽門螺旋桿菌感染胃腺癌上皮細胞(AGS cell line)時,會促使Akt, pAkt移到細胞膜上進行活化,且失去活性的GSK3β會促進β-catenin移轉到細胞核當中並增加Cyclin D1在細胞質的分布。 由本論文實驗結果所推測出來一種可能的磷酸化訊息傳遞路徑 (signal transduction by phosphorylated proteins),或許可以解答這個長久以來一直存在的問題:為何當正常胃部細胞受到幽門螺旋桿菌感染之後會走向癌化 (H. pylori mediated carcinogenesis)。 Helicobacter pylori influences cell proliferation, triggers apoptosis, and contributes to the pathogenesis and severity of gastric injury. However, the specific signaling pathways induced by H. pylori still remain unclear. Since phosphorylation is a key regulatory event in most cellular processes and signaling pathways, we established a phosphoproteomics approach, which is a combination of the techniques of immobilized metal affinity chromatography (IMAC) and LC-ESI/MS/MS, and performed Ingenity Pathway Analysis (IPA) software analysis. These studies suggested that glycogen synthase kinase 3 beta (GSK3β) may be phosphorylated during gastric epithelial cell infected by H. pylori. Both western the confocal microscope data confirmed that H. pylori infection can lead to phosphorylation Akt and GSK3β. This is followed by the release of β-catenin from the GSK3β/β-catenin complex, and the subsequent nuclear translocation of β-catenin that triggers cyclin D1 expression in the cytoplasm. Taken together, these results support the hypothesis that H. pylori activates the PI3K/Akt signaling pathway, resulting in phosphorylation and inhibition of GSK3β, subsequent translocation of β-catenin to the nucleus, and increase in the β-catenin-regulated transcriptional activity. These data suggest, for the first time, that some phosphoproteins may play a role in the pathogenesis of H. pylori infection, including the development of gastric cancer. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42828 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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ntu-98-1.pdf Restricted Access | 5.39 MB | Adobe PDF |
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