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研究生:劉建辰
研究生(外文):Chien-Chen Liu
論文名稱:Alpha-1-antitrypsin (AAT)甲一型抗胰蛋白酵素在胃癌血漿中轉譯後修飾的表現
論文名稱(外文):The expression of post-translational modification ofAlpha-1-antitrypsin in the plasma of gastric cancer
指導教授:林景堉
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:61
中文關鍵詞:甲一型抗胰蛋白酵素胃癌轉譯後修飾
外文關鍵詞:post-translational modificationAlpha-1-antitrypsin
相關次數:
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癌症在國人的十大死因連續三十一年排名第一,在台灣胃癌的死亡率是居癌症死因的第六位,然
而當胃癌在癌症早期就被診斷出來時,其生存率高達95%以上,故一個能早期診斷胃癌的生物標
記是必要的。鑑於此理由, 開發出新的診斷方法能提高診斷胃癌的特異性和敏感度,是迫切之急的
任務。本篇研究主要在探討Alpha-1-Antitrypsin(AAT)甲一型抗胰蛋白酵素在血漿中轉譯後修飾的
表現,以蛋白質體分析策略,找出有意義的轉譯後修飾拿來做為開發檢測胃癌的生物標記。透過
比較健康人及胃癌患者血漿中,蛋白質量(表現量)及質(轉譯後修飾)的變化,找出可以運用
到臨床的生物標記。在本研究中,實驗材料是收集正常人46 位和胃癌病人46 位的血漿檢體。
先進行免疫沉澱法,確認抗體有抓到目標蛋白Alpha-1-Antitrypsin(AAT),再使用一維凝膠電泳
(SDS-PAGE)和西方墨點法(Western blot)探討AAT 在胃癌血漿中的蛋白質表現量;之後,再利用膠
體內水解(In-Gel digestion)的方式.將我們需要的蛋白質區塊在一維凝膠電泳(SDS-PAGE)上切下,
打入奈米級液相層析串連質譜儀(nano-LC/MS/MS)。分析蛋白質以trypsin 水解脢降解成胜肽碎片
所得到的數據,推測胜肽的氨基酸序列。收集質譜資料後加以分析,資料庫比對後找出可能發生
的修飾然後鑑定出轉譯後修飾的位置。實驗結果顯示AAT 的蛋白質表現量在胃癌早期與正常人
相比沒有明顯的差異(P=0.186),且在胃癌晚期與正常人相比也無明顯差異(P=0.091)。此外,AAT
的 ROC curve AUC 只有 0.660,靈敏度和特異性分別為 71.74%和63.04%,得知利用血漿中的
蛋白質表現量無法分辨胃癌早晚期和正常人的差異,沒有明顯的變化。
因此改為探討 AAT 的轉譯後修飾表現。發現 AAT 的胺基酸上有許多新穎性的修飾且倍數差
異在三倍以上包含有methylation(Glu-370、Glu-378、Glu -387 Lys-367)、Hydroxylation(Asn-271、Pro-279、
Asp-280、Lys-283)、Dimethylation (Proline-385)和n-Decanoate(Serine -325)。所以結論是我們可以利用
這些蛋白質轉譯後修飾的差異特性當作診斷胃癌的生物標記並且加以量化比較。

The leading cause of death is contributing to cancer in our country ranked over thirty-one consecutive
years .Gastric cancer is the sixth leading cause of cancer deaths in Taiwan, yet survival rates are over
95% when it is diagnosed at an early stage, the need for biomarkers for early diagnosis can’t be
overemphasized. So, to issue a new diagnostic method can improve the specificity and sensitivity of
diagnosis of gastric cancer is very important. This study was to investigate the translational
modifications (PTM) in plasma performance of Alpha-1-Antitrypsin (AAT). So we utilize proteomic
analysis strategy, by comparing healthy people and patients with gastric cancer plasma protein quality
and qualitative changes can be applied to identify clinical biomarkers.
In the present study, we used some approaches including Immunoprecipitation ,nano-LC-MS/MS ,
western blot to screen AAT biomarker in plasma samples which were obtained from 46patients with
gastric cancer and 46 healthy volunteers. Then we study those statistics and PTMs in order to find out
useful diagnostic tools.
Experimental results indicate that neither early stage gastric cancer group nor late stage gastric cancer
group show no significant differences compared with Normal group. Thus, to use the plasma protein
expression can’t distinguish between cancer and normal differences between early and late cancer stage.
So we change the side to observe the post-translational modifications of AAT performance. The PTMs
data showed there were a lot of PTMs in gastric cancer,including methylation(Glu-370、Glu-378、Glu -387
Lys-367)、Hydroxylation(Asn-271、Pro-279、Asp-280、Lys-283)、HexHexNAc(Threonine-369)、Dimethylation
(Proline-385)and n-Decanoate(Serine -325)。The ROC curve shows that those modifications are perfect
tools to identify gastric cancer。In conclusion,those PTMs will be good biomarkers that we can use
these proteins characteristic differences to screen cancer and quantify it。

第一章、 緒論 ......................................................................................................... 1-11
第一節、 胃癌之流行病學 ..................................................................... 1
第二節、 胃癌的分類 ......................................................................... 1-4
第三節、 胃癌的診斷 ............................................................................. 4
第四節、 胃癌診斷之蛋白質生物標記 ............................................. 4-8
第五節、 胃癌之血漿蛋白質轉譯後修飾分析應用於生物標記開發……9-10
第六節、 胃癌診斷之蛋白質生物標記 ......................................... 10-11
第二章、 研究策略 ............................................................................................... 12-13
第三章、 研究方法與材料 ................................................................................... 14-20
第一節、 樣品蒐集與製備 ................................................................... 14
3-1.1、 胃癌血漿樣品蒐集 ................................................ 14
3-1.2、 胃癌血漿蛋白質濃度定量 .................................... 14
第二節、 西方墨點法(Western blot) ............................................. 14-15
第三節、 免疫沉澱法(Immunoprecipitation, IP) ........................... 15-17
第四節、 質譜分析 ......................................................................... 17-19
3-4.1、 一維凝膠電泳(SDS-PAGE) ................................. 17
3-4.2、 膠體內水解(In-Gel digestion) .......................... 17-18
3-4.3、 奈米級液相層析串聯質譜(nano-LC/MS/MS) .. …18
3-4.4、 資料庫檢索 ............................................................ 19
第五節、 轉譯後修飾定量 ................................................................... 19
第六節、分子模型 ............................................................................... 20
第七節、 統計分析 ............................................................................... 20
第四章、 結果 ....................................................................................................... 21-25
IV
第一節、 西方墨點法分析 ................................................................... 21
第二節、 免疫沉澱法結果 ............................................................. 21-22
第三節、 質譜分析 ............................................................................... 22
第四節、 轉譯後修飾 ..................................................................... 22-23
第五節、 碎片圖分析 ........................................................................... 24
第六節、 分子模型 ............................................................................... 25
第五章、 討論 ....................................................................................................... 26-29
第六章、 結論 ............................................................................................................. 30
第七章、 未來發展 ..................................................................................................... 30
第八章、 參考文獻 ............................................................................................... 55-61

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