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研究生：	陳成欣 Ting, Sing-Sing
論文名稱：	以蛋白質體學及質譜儀分析方法比較在發生腹膜炎前後腹膜透析液中的差異蛋白質表現 A comparative proteomics analysis of peritoneal dialysate before and after the occurrence of peritonitis episode by mass spectrometry
指導教授：	廖寶琦 Liao, Pao-Chi
學位類別：	碩士 Master
系所名稱：	醫學院 - 環境醫學研究所 Department of Environmental and Occupational Health
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	46
中文關鍵詞：	腹膜透析、腹膜透析液、腹膜炎、蛋白質體學
外文關鍵詞：	peritoneal dialysis, peritoneal dialysate, peritonitis, proteomics
相關次數：	點閱：108 下載：6
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腹膜透析是治療末期腎臟病病人的其中一種方式。但腹膜長期接觸高濃度的葡萄糖透析液將會造成結構及功能之變化。至今，腹膜炎仍是腹膜透析的主要併發症，其將加速損傷腹膜及增加其通透性及降低超過濾功能，最終病人因水份堆積導致心臟衰竭的機會增加，或需改換進行血液透析。因此本研究為要更一步瞭解在發生腹膜炎之後，透析液中蛋白質的變化。本研究使用二維電泳結合逆向奈米高效能液相層析電噴灑串聯式質譜儀進行分析，之後利用西方墨點法及酵素免疫分析法進行驗證。此研究共分析12對腹膜炎之前及之後的透析液樣本，總共偵測大約400-600個蛋白點。自這些點中，挑選出136個點進行蛋白酶消化後進行質譜分析。我們總共鑑定出41個高可信度的蛋白質，其中有十個蛋白質在發生腹膜炎之前及之後有顯著差異。此研究主要是要建立發生腹膜炎之前及之後的蛋白質身份之圖譜。這些蛋白並非新的生物指標，但或許能顯示病人是否需要給予藥物治療之狀況，未來亦有可能成為腹膜炎之指標但需要更多深入的研究。

Peritoneal dialysis (PD) is one of the therapeutic options for end-stage renal disease (ESRD). The peritoneal membrane is immersed in a high glucose concentration of the peritoneal dialysate which in long term may cause structural and functional damage to the peritoneal membrane. Peritonitis is one of the major complications of PD which will accelerate the damage of peritoneal membrane by increasing the peritoneal permeability and decrease the ultrafiltration efficiency. Finally it will cause the peritoneal membrane dysfunction and the patient may have fluid overload related complications and have to switch to hemodialysis. Thus, this study aim to enhance our understanding of the protein changes in peritoneal dialysate after the occurrence of peritonitis. In this study, we performed 2DE on 12 pairs of peritoneal dialysate before and after the peritonitis and approximately 400~600 spots were detected. Among these spots, 136 spots were excised, tryptic digested and identified by RP-nano-HPLC-ESI-MS/MS. 41 proteins with high levels of confidence were identified. Among these, ten proteins were significantly differentially expressed in peritoneal dialysate before and after the peritonitis, Western blot and ELISA validation were done. The present study was designed to develop a proteomic map of proteins before and after the peritonitis occurrence. These proteins are not the new biomarkers but they may indicate a situation for possible drug treatment, it may be the predictors of peritonitis which required validation study in future.

Abstract..................................................I
摘要........................................II
誌謝..........................................III
Table of contents...........................IV
List of Figures.................................VI
List of Tables......................................VII
List of Abbreviations..........................VIII
Chapter 1  Introduction
    1.1 Epidemiology of end-stage renal disease(ESRD)...1
    1.2 Peritoneal dialysis(PD).......................1
    1.3 The benefits of PD................2
    1.4 Peritoneal Equilibration Test (PET)........2
    1.5 The physiological changes of peritoneal membrane    during peritonitis.......2
    1.6 Studies of PD-related peritonitis........3
    1.7 The aim of this study.................4
Chapter 2  Material and Methods
    2.1 Samples collection...................5
    2.2 Total protein concentration assay.........5
    2.3 Protein precipitation.................6
    2.4 Two dimensional gel electrophoresis (2DE)
       2.4.1 First dimension: Isoelectric focusing (IEF)...7
       2.4.2 Second dimension: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).....8
    2.5 Silver stain............................9
    2.6 Image acquisition and analysis.............11
    2.7 Tryptic in-gel digestion...........11
    2.8 Protein identification by reverse-phase nano high performance liqiud chromatography-electrospray ionization-tandem mass spectrometry(RP-nano-HPLC-ESI-MS/MS).........12
    2.9 Western blotting.....................14
    2.10 Enzyme-linked Immunosorbent assay (ELISA).......15
Chapter 3  Results
    3.1 Demographic data of patients (n=12)........16
    3.2 2DE protein profiling of peritoneal dialysate before and after the occurrence of peritonitis........16
    3.3 Cellular location of the identified proteins after peritonitis episode.............34
    3.4 Biological processes and molecular function of identified proteins...35
    3.5 Validation of 2DE analysis using Western blot...36
    3.6 Validation of 2DE analysis using ELISA.....37
Chapter 4  Discussion
    4.1 Differential proteins identified by RP-nano-HPLC-ESI-MS/MS........38
    4.2 Acute phase/immune response-related proteins....39
    4.3 Binding/transport-related proteins......40
    4.4 Blood coagulation/hemostasis-related proteins...41
    4.5 Study limitations.....................42
Conclusions........................................43
References..........................................44

                                    

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校內：2016-02-05公開
校外：2016-02-05公開

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