先前的實驗已使用二維螢光差異表現蛋白質電泳配合基質輔助雷射脫附游離質譜法找出正常成人經過太極拳運動後在血清上表現的蛋白質差異(Yang & Chang, et al. Clin Chem 56:127, 2010 )。但是血清中高豐富度的蛋白質同時也掩蓋掉了低豐富度蛋白質的表現，在二維電泳上那些低豐富度或微量的蛋白質不易被發現，但這些微量蛋白或低豐富度蛋白質在生理學上卻扮演了重要的角色。因此我們尋找其他替代方法來改善這個現象，我們知道想增加蛋白質的表現就必須多增加幾個分離的步驟。在經過多方嘗試後，選用了可抓除血清內十四種高豐富度蛋白質的親和性管柱，藉由其快速液相層析儀自動純化分離蛋白質，得到的低豐富度蛋白質；再藉由一維凝膠電泳將蛋白質依照分子量大小做第一次的分離，再者利用樣品與溶液極性不同的奈米液相層析儀做第二次的分離。最後將分離完的樣品利用電噴灑游離法，做一次質譜及二次質譜的掃描。得到的結果經軟體分析其功能，區分三組；分別為正常成人運動前後、糖尿病人運動前後及正常成人與糖尿病人運動前。然後從每組的第一個最有意義的路徑裡挑出蛋白質可以用酵素連結免疫吸附法做確認者。結果發現糖尿病人在運動前雙胜肽酶4 (Dipeptidyl peptidase 4；DPP4)和嗜中性白血球明膠相關性脂質運載蛋白(Neutrophil gelatinase-associated lipocalin；NGAL)顯著高於正常成人(P值分別為0.011和小於0.001)，而泌乳激素誘導蛋白(Prolactin-inducible protein；PIP)則是正常成人運動前高於糖尿病人運動前(P值為0.042)。文獻指出抑制DPP4作用在第2型糖尿病人上會降低血糖值並提高免疫力；另外NGAL也證實可拿來做早期診斷急性腎損傷的指標。我們研究發現糖尿病人兩者皆比正常人高，值得進一步研究哪些運動可以降低DPP4和NGAL。而在有效去複雜化蛋白質下，確實能得到不同的低豐富度蛋白質，也可能找到代謝與免疫指標，另藉由太極拳運動前後的模式改變也可能找到運動促進免疫的指標。

We have previously used a two-dimensional fluorescence difference gel electrophoresis protein expression with matrix-assisted laser desorption ionization of mass spectrometry to identify the serum proteomic profiles before and after the Tai Chi exercise in normal adults (Yang & Chang, et al. Clin Chem 56:127, 2010). However, the high abundant serum proteins in seyal samples might interfere the discovery of low abundance proteins in two-dimensional electrophoresis, but these low abundant proteins may play an important role on human physiology. Therefore, we looked for another way to resolve this complex issue. After multiple attempts, we chose a commercial affinity column to exclude 14 kinds of high-abundance proteins before analyses of serum proteomic displays. This column could be fit into a fast liquid chromatography separation of purified proteins and eluted for low abundant proteins. The low abundant proteins were first expressed by one-dimensional gel electrophoresis of proteins followed by a series of gel cut down for in-gel digestion by trypsin and subject to nano-liquid chromatography with electrospray ionization tandem mass spectrometry analysis (nano-LC ESI MS/MS ). The results obtained by software analysis were subject to its functional pathways analysis. We analysed 3 comparisons of the protein displays including differences between normal adults before and after exercise, differences of normal adults and diabetic patients before and after exercise. Experiments were next performed to validate the most significant difference of proteins between each category by enzyme-linked immunoassay. Results showed that dipeptidyl peptidase 4 (DPP4) and neutrophil gelatinase-associated lipocalin (NGAL) proteins were significantly higher in diabetic patients than in normal adults ( P values were 0.011 and less than 0.001), while the prolactin-inducible protein (PIP) was higher in normal adults than in diabetic patients after exercise (P value of 0.042). To our knowledge, decrease of DPP4 in type 2 diabetes has been shown to reduce blood sugar and improve the immunity; and NGAL has been confirmed to be an indicator for early diagnosis of acute kidney injury. Therefore, we have identified certain functional proteomic markers in normal and diabetic patients after Tai Chi exercise. This study model with exclusion of high-abundance serum proteins is a useful mode for identifying immune and metabolic marker with and without.

目 錄
誌謝................................................................................................................................... i
中文摘要........................................................................................................................... ii
英文摘要........................................................................................................................... iii
圖次................................................................................................................................... vii
表次................................................................................................................................... ix
第一章 緒論...................................................................................................................... 1
1.1 前言............................................................................................................................ 1
1.2 糖尿病分類、診斷及治療........................................................................................ 3
1.3 奈米液相層析電噴灑串聯式質譜儀應用(Nano liquid chromatography
electrospray ionization tandem mass spectrometry；Nano LC-ESI MS/MS).... 3
第二章 實驗材料與方法.................................................................................................. 5
2.1 實驗材料.................................................................................................................... 5
2.2 實驗設備及軟體........................................................................................................ 7
2.2.1 設備......................................................................................................................... 7
2.2.2 軟體......................................................................................................................... 7
2.3 實驗架構.................................................................................................................... 8
2.4 本實驗流程圖............................................................................................................ 9
2.5 實驗方法................................................................................................................... 10
2.5.1 血液收集................................................................................................................ 10
2.5.2 太極拳運動(Tai Chi exercise)............................................................................ 10
2.5.3 血中十四種高豐富度蛋白質去除(Human-14 protein depletion).................. 10
2.5.4 蛋白質定量............................................................................................................ 11
2.5.5 一維凝膠電泳(SDS-PAGE)及染色..................................................................... 11
2.5.6 上機前處理-膠內蛋白質酵素水解(In-gel digestion)....................................... 12
2.5.7 奈米液相層析儀(碳十八管柱)偵測條件............................................................. 13
2.5.8 電噴灑離子化串連式質譜儀偵測條件................................................................ 13
2.5.9 差異蛋白質線上搜尋歸類分析............................................................................ 14
2.5.10 以酵素連結免疫吸附法(Enzyme-linked immunosorbent
assay, ELISA ) 確認蛋白質差異.................................................................................. 14
2.5.10.1 ELISA 方法定量泌乳激素誘導蛋白
(Prolactin-inducible protein；PIP)............................................................................. 14
2.5.10.2 ELISA 方法定量雙胜肽酶4 (Dipeptidyl peptidase 4；DPP4)................. 15
2.5.10.3 ELISA 方法定量定量甘露糖結合凝集素絲氨酸
蛋白酶1 (Mannan-binding lectin serine protease 1 ；MASP1)............................ 16
2.5.10.4 ELISA 方法定量硒蛋白質P (Selenoprotein P1, Plasma；SEPP1)....... 16
2.5.10.5 ELISA 方法定量嗜中性白血球明膠相關性脂質運載蛋白
(Neutrophil gelatinase-associated lipocalin ；NGAL).......................................... 17
第三章 結果.................................................................................................................... 18
3.1 血中十四種高豐富度蛋白質去除效果 (Human-14 protein depletion)............ 18
3.2 一維凝膠電泳 (SDS-PAGE)................................................................................... 18
3.3 蛋白質鑑定結果....................................................................................................... 18
3.4 以酵素連結免疫吸附法確認蛋白質濃度............................................................... 20
第四章 討論與未來展望................................................................................................. 21
4.1 蛋白質鑑定結果....................................................................................................... 21
4.2 未來展望................................................................................................................... 22
參考文獻.......................................................................................................................... 69
附錄.................................................................................................................................. 74

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