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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周綠蘋 | |
dc.contributor.author | Shu-Yu Lee | en |
dc.contributor.author | 李書宇 | zh_TW |
dc.date.accessioned | 2021-06-12T18:08:34Z | - |
dc.date.available | 2016-10-05 | |
dc.date.copyright | 2011-10-05 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-08 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27530 | - |
dc.description.abstract | 胃癌在全球癌症死因中名列第二,每年約有一百萬人死於此疾病。造成胃癌的主要原因除了飲食及遺傳因子外,幽門螺旋菌(Helicobacter pylori)的感染也是其中一個重要因素。過去研究已證實,幽門螺旋菌的感染的確與胃癌的發生息息相關,然其引發胃癌的詳細機制仍不清楚。本實驗室使用人類胃腺癌上皮細胞(AGS cell line)和幽門螺旋菌作為生物材料,並與國家衛生研究院褚志斌老師實驗室合作,利用微西方點墨陣列(microwestern arrays)分析未感染、及經幽門螺旋菌感染的胃腺癌上皮細胞,其蛋白質表現或轉譯後修飾的改變;希望能藉此對幽門螺旋菌引發胃癌的機制作有系統的瞭解。實驗結果顯示,在幽門螺旋菌所導致的胃癌中,激酶 Akt 的活化可能扮演重要角色。因此我們希望藉由質譜蛋白質體學方法(mass spectrometry-based proteomic approach),進一步找出激酶 Akt 的下游受質。首先使胃腺癌上皮細胞大量表現激酶 Akt,並利用免疫共沉澱法(co-immunoprecipitation)分離出與激酶 Akt 有交互作用的蛋白質。經由液相層析串聯質譜儀(liquid chromatography tandem mass spectrometry)鑑定以及蛋白質資料庫搜尋比對,我們共找到 64 個與 Akt 有交互作用的蛋白質。將這些蛋白質送入訊息傳導資料庫(Ingenuity Systems Pathway Analysis)分析其可能參與的訊息傳導路徑;結果顯示,激酶 Akt 能透過磷酸化 FOXO3A、Caspase-9、BAD、p21 來調控下游宿主反應。進一步使用西方點墨法(western blot)驗證,我們發現在幽門螺旋菌感染下,細胞中 FOXO3A、Caspase-9、BAD、p21 蛋白質,其磷酸化的確有上升的情形。接著使用 LY294002 處理細胞,使激酶 Akt 活性被抑制,並觀察 FOXO3A、Caspase-9、BAD、p21 的磷酸化是否也隨之減少;結果發現 FOXO3A 在 Ser 253 位置的磷酸化的確有降低的情形。而過去研究也指出, FOXO3A 的 Ser 253 位置被激酶 Akt 磷酸化後,將促進細胞存活(cell survival)。未來可望針對這些結果做進一步探討,以期對激酶 Akt 所調控的幽門螺旋菌致病機制有更深入的瞭解。 | zh_TW |
dc.description.abstract | Gastric cancer is the second leading cause of cancer death worldwide. The main risk factors for gastric cancer include dietary factors, host genetics and H. pylori (Helicobacter pylori) infection. In 1994, H. pylori was classified as a type I carcinogen by IARC (International Agency for Research on Cancer). Bacterial-induced chronic inflammation results in 1 ~ 3% of infected patients transforming to gastric cancer. We chose AGS cell line and H. pylori as biomaterials, and cooperated with Dr. Chuu’s Lab for the MWA (microwestern arrays) technique. Comparing the proteome of non-infected and H. pylori-infected AGS cells with MWAs, we found that phosphorylated Thr 308 and Ser 473 of Akt were elevated in response to H. pylori stimulation. Previous studies have shown that phosphorylated Thr 308 and Ser 473 of Akt can cause its activation and downstream targets’ phosphorylation. After overexpressing Akt in AGS cells and co-IP (co-immunoprecipitating) the whole complexes, we used LC-MS/MS (liquid chromatography tandem mass spectrometry) to identify the Akt interactome. Deducting out of the non-specific binding proteins, we identified 64 potential Akt interacting proteins. Analyzing the 64 identified proteins with IPA (Ingenuity Systems Pathway Analysis), we found the possible Akt downstream targets, including FOXO3A, Caspase-9, BAD and p21, may involve in anti-apoptotic pathways. We further validated these possible Akt downstream targets with western blots and observed the phosphorylation level of FOXO3A, Caspase-9, BAD and p21 were elevated in response to H. pylori stimulation. However, after treating the inhibitor LY294002, which inhibits Akt activity, only phosphorylated Ser 253 of FOXO3A decreased. Previous studies have shown that phosphorylated Ser 253 of FOXO3A can cause its cytosolic retention and promote cell survival. In the future, we will further validate these phosphorylation regulations and their physiological roles. This information may help to demonstrate more comprehensive pathogenic mechanisms of Akt signaling in H. pylori-induced gastric cancer development. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:08:34Z (GMT). No. of bitstreams: 1 ntu-100-R98442018-1.pdf: 118262754 bytes, checksum: 9f50e0fa88db730fd41a374c95625eb8 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 謝誌....................................................................................................................................i
摘要...................................................................................................................................ii Abstract............................................................................................................................iii 縮寫..................................................................................................................................iv 第一章 導論.....................................................................................................................1 1.1 胃癌................................................................................................................1 1.2 幽門螺旋菌....................................................................................................3 1.3 蛋白質體學....................................................................................................6 1.4 激酶 Akt.......................................................................................................10 1.5 研究動機......................................................................................................12 第二章 實驗材料...........................................................................................................14 2.1 幽門螺旋菌菌株..........................................................................................14 2.2 胃腺癌細胞株..............................................................................................14 2.3 細胞培養液及試劑......................................................................................14 2.4 儀器及裝置..................................................................................................14 2.5 酵素..............................................................................................................15 2.6 藥品與試劑組..............................................................................................15 2.7 軟體與資料庫..............................................................................................16 第三章 實驗方法...........................................................................................................17 3.1 幽門螺旋菌與胃腺癌上皮細胞培養..........................................................17 3.2 以幽門螺旋菌感染胃腺癌上皮細胞..........................................................18 3.3 基因轉染......................................................................................................18 3.4 以 LY294002 處理胃腺癌上皮細胞...........................................................20 3.5 微西方點墨陣列..........................................................................................21 3.6 細胞蛋白質抽取與定量..............................................................................23 3.7 十二烷基磺酸鈉-聚丙烯醯胺膠體電泳.....................................................24 3.8 西方點墨法..................................................................................................26 3.9 免疫共沉澱..................................................................................................27 3.10 蛋白質鑑定................................................................................................28 3.11 資料庫分析................................................................................................30 第四章 實驗結果...........................................................................................................31 4.1 分析幽門螺旋菌感染胃腺癌上皮細胞後蛋白質磷酸化修飾情形..........31 4.2 分析胃腺癌上皮細胞中激酶 Akt 之交互作用體......................................32 4.3 以 IPA 資料庫分析訊息傳導路徑..............................................................33 4.4 以西方點墨法驗證 IPA 資料庫分析結果..................................................34 第五章 討論...................................................................................................................35 5.1 微西方點墨陣列分析..................................................................................35 5.2 激酶 Akt 交互作用體之分析......................................................................36 5.3 結語及未來展望..........................................................................................39 第六章 參考文獻...........................................................................................................41 第七章 圖表與說明.......................................................................................................49 附錄.................................................................................................................................60 | |
dc.language.iso | zh-TW | |
dc.title | 利用蛋白質體技術鑑定人類胃腺癌上皮細胞中激酶 Akt 之交互作用體 | zh_TW |
dc.title | Proteomic Approach to Identify the Akt Interactome in the Human Gastric Adenocarcinoma Epithelial Cell Line AGS | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 繆希椿,褚志斌,蔡孟勳 | |
dc.subject.keyword | 胃癌,幽門螺旋菌,激酶,Akt,蛋白質體學,訊息傳導, | zh_TW |
dc.subject.keyword | gastric cancer,Helicobacter pylori,Akt,proteomics,signal transduction, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-09 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
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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