血液透析被廣泛應用在腎衰竭病人的治療，目前已知血液透析會對病人產生很大的氧化壓力。為了瞭解哪些蛋白質會吸附在透析管上，且蛋白質是否更被氧化，本實驗收集了五位洗腎病人的透析管並將吸附在人工合成膜的蛋白質萃取出來。利用蛋白質二維電泳法和質譜鑑定去分析定義出吸附在透析管的蛋白質，以及氧化偵測之西方墨點法套組去偵詴吸附蛋白質的氧化狀況。結果顯示在二維電泳膠圖中更一百五十三個膠點，定義出二十種會吸附在透析膜上的蛋白質，其中包含更Vsm-RhoGEF 、Fibroblast growth factor 23、 Prothrombin、Glomulin和 Nucleobindin-1。而在氧化測詴結果中透析管吸附蛋白的氧化包含更MAP4K3 protein和 Sulfiredoxin-1。透析管蛋白的氧化現象比血清蛋白來的高，而這些蛋白氧化的程度可能與病人發炎指數( CRP level )更很高的相關性。本實驗首次萃取出臨床上洗腎病人透析管上吸附的蛋白質，並利用二維電泳技術和質譜分析定義出二十種會吸附在透析管上的蛋白質以及五種被氧化的白質，還更蛋白質氧化現象和病人的發炎指標的相關性。

Hemodialysis is widely used for kidney failure patients, it is well known that oxidative stress is induced during hemodialysis process. To figure out what kind of proteins may adhere to the hemodialysis tube, and were those proteins oxidized during dialysis process. In this study, proteins adhere to hemodialysis membrane are eluted and examined by protein two-dimensional gel electrophoresis and MALDI-TOF technique. The two-dimensional gel electrophoresis results demonstrate 153 proteins binding to hemodialysis tube including Vsm-Rho GEF, Fibroblast growth factor 23, Prothrombin, Glomulin and Nucleobindin-1. The oxidation detection shows that some tube binding proteins are oxidized including MAP4K3 protein and Sulfiredoxin-1, and the oxidation level of hemodialysis tube binding proteins are higher then serum proteins. In conclusion, we find out 20 novel proteins which bind to hemodialysis tube and 5 novel proteins which oxidized during hemodialysis. And protein oxidation level was related to inflammation where high CRP levels were detected.

Content
中文摘要 1
英文摘要 2
英文縮寫表 3
壹、前言 4
一.血液透析管吸附蛋白質之分析與重要性 4
1.1腎臟與泌尿系統解剖圖.解剖圖 5
1.2腎臟的基本功能 5
1.3病理特性與分期 6
1.4腎衰竭之病因學 7
1.4.1慢性腎絲球炎 7
1.4.2多囊性腎病變 8
1.4.3糖尿病腎病變 8
1.4.4間質性腎炎 10
1.5末期腎衰竭之治療 11
1.5.1血液透析 11
1.5.2 腹膜透析 11
1.5.3腎移植 12
1.6血液透析與氧化壓力 13
貳、實驗流程(Experiment flowchart)…14
參、材料與方法(Materials and Methods)…15
一.血液透析病人之血液與透析管取樣 15
1.檢體來源 15
2.血液取樣與蛋白質萃取 15
3.透析管取樣 15
二.透析管吸附蛋白之萃取 16
1.蛋白質之萃取與濃縮 16
2.TCA/Acetone 沉澱 17
3.蛋白質定量 17
三.二維電泳法 18
1.Isoelectric focusing electrophoresis 聚電焦電泳 18
2.Immobiline DryStrip Equilibration 19
3.SDS-PAGE聚丙烯酼胺膠體電泳 20
4.Coomassie brilliant blue R-250 stain 21
5. In gel digestion 22
5.1 清洗(Washing) 22
5.2 Coomassie brilliant blue R-250退染 22
5.3 Reduction/Alkylation 22
5.4 Digestion/Extraction 23
5.5 MALDI-TOF質譜操作 24
5.6 使用ProteinProspector之MS-Fit來辦認為之的蛋白質 24
四.氧化蛋白測定 25
肆.實驗結果 28
一.血液透析病人檢體之分析結果 28
二.二維電泳膠片之分析結果 29
三.MALDI-TOF質譜分析與蛋白質資料庫比對 29
3.1 MS質譜分析 29
3.2差異性蛋白質身份鑑定 30
四、透析管吸附蛋白質之氧化測詴 31
五、透析管吸附之氧化蛋白身分鑑定 31
六、血液透析氧化與發炎反應之相關性 32
伍.實驗討論(Discussion) 33
一.透析膜結合蛋白之二維電泳實驗討論 33
二.病人血清與透析管吸附蛋白之蛋白質氧化測詴實驗討論 34
三.透析管吸附蛋白與發炎反應相關性 36
四.差異性蛋白質介紹與討論 37
陸、結論 46
柒.參考文獻 47
捌.圖與表(Figures & Tables) 50
表 一.血液透析病患基本資料與相關臨床數據一覽表 50
圖一. 透析管吸附蛋白之二維電泳膠圖 51
圖二. 透析管吸附蛋白之選取膠點圖 52
圖三. 血清與透析管吸附蛋白質之一維電泳圖與氧化測詴 53
圖四.透析管吸附蛋白氧化測詴之量化圖 54
表二、透析管吸附蛋白氧化與C-reactive protein 54
附圖一~附圖二十五. 透析管吸附蛋白質經過Trypsin分解片段的質譜分析 56
附表一~附表二十五. 透析管吸附蛋白質之MS peaks match coverage percentage 56

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