臺灣博碩士論文加值系統

中文摘要
本研究目的為利用二維電泳法 (Two-dimensional electrophoresis ; 2-DE) 以找出ENU (ethyl-nitrosourea)突變小鼠表現差異的蛋白質點並進行質譜分析，以瞭解突變小鼠短鏈脂肪酸代謝異常原因，並且希望能應用此一模式動物於脂肪酸的代謝研究及探討人類相關臨床疾病。我們利用ENU注射動物方法藉以誘發大量基因突變小鼠的產生，經由繁殖配對至第三代後，再以串聯式質譜儀篩得血漿短鏈脂肪酸 (C4-OH) 代謝異常突變小鼠。實驗小鼠 (C57BL/6J) 分為正常小鼠及血漿C4-OH含量較正常小鼠高4倍標準差的ENU突變小鼠，兩組小鼠分別犧牲後取其肝臟、肌肉組織，進行粒線體蛋白質萃取後以SDS-PAGE進行一維電泳，然而突變小鼠和正常小鼠粒線體蛋白質表現量並無差異。因此，更進一步利用二維電泳法進行突變小鼠和正常小鼠粒線體蛋白質表現的比較。研究結果顯示突變小鼠和正常小鼠的肌肉粒線體存有差異的蛋白質點，經由介質輔助雷射脫附游離-飛行式質譜儀 (Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; MALDI-TOF) 鑑定出差異蛋白質點的身份。在突變小鼠表現量增加的蛋白質為磷酸化的myosin regulatory light chain 2、tropomysin 1 α chain、 myosin light chain 1，表現量減少的蛋白質為非磷酸化的myosin regulatory light chain 2、calsequestrin-1 precursor、adenylate kinase isoenzyme 1、ATP synthase D chain。然而這些蛋白質表現的消長是否與突變鼠體內C4-OH含量上升有關，仍有待更進一步的研究加以驗證。
關鍵詞：致突變劑 (ethyl-nitrosourea; ENU)、串聯質譜儀、短鏈脂肪酸、二維電泳法(Two-dimensional electrophoresis ; 2-DE)、介質輔助雷射脫附游離-飛行式質譜儀(Matrix-assisted laser desorption / ionization time-of-flight mass Spectrometry; MALDI-TOF)

Abstract
The purpose of this study was to develop a 2-DE coupling with MALDI-TOF analysis to find the differential proteins in ENU mouse which could explore the cause of genetic defect of short chain fatty acid in mice. We hope that the C4-OH animal model can provide us to investigate fatty acid metabolism related to human genetic disease. We generated ENU- mutagenized mouse and applied to MS/MS tandem mass spectrometry to screen third generation progeny of ENU-mutagenized mouse for abnormalities in the pathway of fatty acid metabolism. The short chain fatty acid (C4-OH) level in ENU mice was three to four folds than normal mice. After protein evaluation, there was no differential protein in mitochondria fraction between normal and ENU mice by SDS-PAGE. Surprisingly, there were eleven differential proteins between normal and ENU mice in muscle in 2-DE approach. These protein spots were applied to MALDI-TOF for futher identification. In results, ENU mouse showed higher protein expressions in phosphorylated myosin regulatory light chain 2, myosin light chain 1 and tropomyosin 1αchain and lower expressions in myosin regulatory light chain 2, adenylate kinase isoenzyme, ATP synthase D chain and calsequestrin-1 precursor in ENU mouse to normal control. These differential proteins expression whether involve to increase C4-OH value in mice needs further study to verify.
Key words：Two-dimensional electrophoresis, ethyl-nitrosourea, C4-OH, MS/MS tandem mass, MALDI-TOF

目錄
中文摘要…………………………………………………………………Ⅰ
英文摘要…………………………………………………………………Ⅲ
致謝………………………………………………………………………Ⅳ
目錄 …………………………………………………………………… Ⅶ
圖目次…………………………………………………………………. Ⅸ
表目次…………………………………………………………………. Ⅹ
縮寫表……………………………………………………………… .. XI
第一章 緒論…………………………………………………………… 1
第一節 研究動機與目的…………………………………………. 1
第二章 文獻回顧……………………………………………………… 2
第一節 ENU突變劑的致突變力…………………………………. 2
第二節 脂肪酸代謝及相關性的代謝疾病.………………………. 4
第三節 蛋白質體學法的應用…………………………………….. 7
第三章 實驗材料與方法……………………………………………… 8
第一節 實驗流程…………………………………………………. 8
第二節 動物來源及飼養環境……………………………………. 8
第三節 串聯式質譜儀篩選………………………………………… 9
第四節 實驗分組…………………………………………………… 9
第五節 粒線體萃取……………………………………………… 10
第六節 總蛋白質含量測定……………………………………… 12
第七節 膠體電泳 ………………………………………………….. 13
第八節 二維電泳 ………………………………………………….. 16
第四章 結果……………………………………………………………26
第一節 串聯式質譜儀脂肪酸代謝物(C4-OH)含量………………. 26
第二節 SDS-PAGE實驗結果……………………………………. 26
第三節 二維電泳實驗結果……………………………………….. 26
第四節 蛋白質身份鑑定結果……………………………………...27
第五章 討論…………………………………………………………..29
第一節 Adenylate kinase isoenzyme………………………………. 30
第二節 ATP synthase D chain……………………………………… 32
第三節 Muscle contraction related proteins…………………………33
第六章 結論…………………………………………………………. 35
第七章 參考文獻……………………………………………………..36
附錄一………………………………………………………………… 61

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