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研究生:歐繡碧
研究生(外文):Ou, hsiupi
論文名稱:人類臍帶間質幹細胞治療肝纖維化小鼠的定量蛋白質體學研究
論文名稱(外文):Study of quantitative proteomics in reduction of mouse liver fibrosis by transplanting human umbilical cord mesenchymal stem cells
指導教授:黃芳正
口試委員:戴嘉慧蕭長春黃芳正
口試日期:2012-07-23
學位類別:碩士
校院名稱:正修科技大學
系所名稱:化工與材料工程研究所
學門:民生學門
學類:美容學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:130
中文關鍵詞:人類臍帶間質幹細胞肝纖維化
外文關鍵詞:ICPL
相關次數:
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人類臍帶間質幹細胞(Humn Wharton’s jelly Mesenchymal stem cell, hWJ-MSC)是分離自新生兒臍帶基質中的細胞,本實驗室先前證實將玻尿酸包覆hWJ-MSC移植至肝纖維化小鼠肝臟薄壁,可明顯改善由四氯化碳所造成的小鼠肝纖維化。
同位素編碼的蛋白質標籤(Isotope-coded protein label,ICPL) 適用於任何蛋白質樣品。選用ICPL的標籤方式一次可同時標籤到四個不同的蛋白質樣品,被標定的樣品做等比例混合後,可以減少其他分離步驟所帶來的樣品複雜度,而此方法也可減少低豐度的蛋白質損失。
本實驗利用上述實驗所得之肝臟組織,使用ICPL的方式標定,配合GeLC-ESI MS/MS做蛋白質鑑定與定量之比較,討論正常小鼠 (ICPL 0)、肝纖維化小鼠 (ICPL 6)與移植WJMSC的病鼠 (ICPL 10,治療組),三者間的蛋白質體變化。
重覆兩次的實驗,第一次得到760個蛋白質,第二次得到719個蛋白質,重覆分析到的蛋白質共571個;另外挑選出高水準的蛋白質 (ICPL peptides≧ 2,CV%< 50),第一次的資料中有261個,第二次的資料中有195個,重覆分析到的蛋白質共有194個。
將這些資料送到MetaCore生物資訊分析平台分析,經查尋相關文獻,與本實驗資料做比對;得到在肝纖維化的組別中胺基酸代謝功能、藥物代謝和脂肪酸代謝功能等有關的許多蛋白質都有相同下降的趨勢,可能是肝纖維化的生化指標 (Biomarker),其中在本實驗治療組的數據中,胺基酸代謝功能(Otc、Haao、Got1、Bckdha、Csad、Glyat、Fthfd、Gpt、Aldh6a1) 有關的9個蛋白質與藥物代謝功能(Ephx2、Bdh、Cyp2f2、Cyp2e1、Cyp1a2、Cyp2a12、Adh4、Aldhla1) 有關的8個蛋白質,和脂肪酸代謝功能(Apoe、Acox、Hadhb) 有關的3個蛋白質及其他功能蛋白質(Rgn、Rps15a、Comt、Ak2) 有關的4個蛋白質,經WJMSC治療後都有回升的趨勢,顯示經由移植WJMSC+HA之後的肝纖維化小鼠部分肝功能有回復的情況,而指出這些蛋白質可能與肝纖維化的修復有關聯,本實驗室未來將繼續探討這些蛋白質是否能治療肝纖維化。

Human umbilical cord mesenchymal stem cells isolated from neonatal umbilical cord matrix cells, our laboratory previously confirmed that hyaluronic acid coated hWJ-MSC transplanted into mice with liver fibrosis liver thin-walled, the significant improvement liver fibrosis caused by carbon tetrachloride.
The isotope-coded protein labels be applied to any protein sample. The selection of ICPL label once the labels to the four different protein samples, calibration samples do mixed in proportion to other separation steps can reduce sample complexity, this method can also be reduced to low abundance protein loss.
In this study, the above experimental results of the liver tissue, using the ICPL calibration to do with the GeLC-ESI MS / MS protein identification and quantitative comparison and discussion among normal mice, mice with liver fibrosis and diseased mice transplanted WJMSC proteome changes.
Experiment repeated twice, first to 760 protein, 719 proteins in the second, repeated analysis of protein 571; other selected high-quality protein (ICPL peptides ≧ 2, CV% <50,), the first data 261 and 195 of the second information, repeat protein analysis to a total of 194.
These data to the the MetaCore bioinformatics analysis platform analysis, the search for relevant literature, with the experimental data of amino acid metabolism, drug metabolism and fatty acid metabolism in the liver fibrosis group comparison; get manyproteins have the same downward trend, may be a biochemical indicator of liver fibrosis (Biomarker), as shown in Table; which data in the experimental treatment group, amino acid metabolism (Otc, Haao, Got1, Bckdha, Csad, Glyat, Fthfd, Gpt, Aldh6a1) 9 protein and drug metabolism (Ephx2 Bdh, Cyp2f2, Cyp2e1, Cyp1a2, Cyp2a12, Adh4 Aldhla1) 8 protein, and fatty acid metabolism (Apoe, Acox, Hadhb)3 protein and other functional proteins (Rgn Rps15a Comt, Ak2) 4 protein recovery after by WJMSC treatment has trend to display through the transplant WJMSC + HA after the mice with liver fibrosis part of the liver functionreply, but pointed out that these proteins may be associated with repair of liver fibrosis, the laboratory will continue to explore whether these proteins can be the treatment of liver fibrosis.

致謝 I
摘要 II
Abstract IV
目錄 VI
圖目錄 VIII
表目錄 XI
第一章 緒論 1
1-1人類臍帶間質幹細胞(hWJ-MSC) 1
1-2肝功能簡介及肝纖維化成因 3
1-3蛋白質的鑑定 5
1-4同位素編碼的蛋白質標籤 (Isotope-coded protein label ,ICPL) 7
1-5一維膠體電泳 10
1-6奈米高效能液相層析結合電噴灑串聯式質譜儀 (nano HPLC-ESI MS/MS) 11
1-6-1電噴灑離子化原理 12
1-6-2離子阱 14
1-7研究動機及目標 15
第二章 實驗材料及方法 16
2-1試劑及材料 16
2-2實驗儀器及軟體 17
2-2-1儀器 17
2-2-2軟體 18
2-3實驗流程 19
2-4實驗方法 21
2-4-1蛋白質萃取及定量 21
2-4-2 同位素標記蛋白質標籤(ICPL)方法 22
2-4-3 膠體電泳(SDS PAGE) 24
2-4-4 膠中消化反應(Ingel Digestion) 26
2-4-5質譜鑑定(Nano LC-ESI MS/MS) 28
2-4-6 蛋白質鑑定分析 32
2-4-7數據整理 37
第三章 實驗結果與討論 39
3-1 小鼠肝臟組織染色 39
3-2 蛋白質鑑定結果 39
3-2-1 高可信度蛋白質篩選結果 40
3-2-2 高可信度且高再現性蛋白質篩選結果 42
3-3 MetaCore 分析結果 43
3-3-1高可信度蛋白質生物資訊分析 43
3-3-2高可信度且高再現性蛋白質生物資訊分析 49
3-4 肝纖維化蛋白質的分析比對 56
第四章 結論及未來展望 61
第五章 參考文獻 63
第六章 附錄 67


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