臺灣博碩士論文加值系統

隨著人類的生活水平的提升、醫療及科技的進步與知識的普及，使得人類壽命愈來愈長，因而老化相關的疾病也開始受到廣泛重視。根據哈曼博士的定義，認為隨著年齡的增加，細胞或組織的傷害累積，導致疾病或死亡的發生，這種現象即稱為老化。目前應用於老化研究之模式生物很多，包含酵母菌、線蟲、果蠅、魚、小鼠與大鼠等；其中在脊椎動物種類最豐富、老化型態最多元的魚類是極佳的選擇。本論文嘗試以「香魚 (ayu, Plecoglossus altivelis)」作為研究對象。香魚的生命週期短，約只有一年，且會在產卵後的一個月左右快速死亡。然而生殖時期是個體生理狀況最高峰的時期，但又要在短短一個月內關閉生理系統造成死亡；因此如能找出香魚快速老化死亡的調控機制，將有助於了解老化的發生。而由於肝臟被認為是承受大量生理壓力的器官，因此本論文便以香魚的肝臟組織為實驗材料，探討其中可能作為老化指標的目標蛋白質。
實驗方法是先將魚隻依雌雄分組，並再細分產卵前 (before spawning)、產卵中 (during spawning)、產卵後 (after spawning) 三個不同時期收取，共計六個群組。以蛋白質體學為分析平台，萃取魚隻之肝臟蛋白質後進行二維電泳分析，得到其肝臟蛋白組成圖譜。擷取膠片影像後接著使用Prodigy SameSpots分析軟體，比對在各時期表現量有顯著變化的蛋白質。將這些蛋白質點挖取並進行膠體內胰蛋白酶水解後，以LC-ESI-Q-TOF MS/MS串聯式質譜儀分析，並藉由生物資訊學軟體Mascot從資料庫中鑑定其身分。
目前共鑑定出九種蛋白質，其中有三種蛋白質的表現量上升者，分別為Betaine-homocysteine S-methyltransferase 1 (BHMT-1)、Triosephosphate isomerase、Glyceraldehyde-3-phosphate dehydrogenase (G3PDH)；六種蛋白質的表現量下降者分別為94 kD glucose-regulated protein (Grp94, Hsp90)、78 kD glucose-regulated protein (Grp78, Hsp70)、Apolipoprotein A-I (Apo A-I)、Tumor necrosis factor receptor、Aldehyde dehydrogenase、Phosphoethanolaminemethyltransferase。並鎖定目前鑑定出的關鍵蛋白質，以即時定量式PCR (q-PCR) 輔助驗證其mRNA表現量變化，並評估其可能作為老化相關指標之潛力。根據上述蛋白質的生理活性，我們歸納發現其與脂質代謝、醣類代謝、心血管疾病、蛋白質摺疊錯誤導致的疾病有關，未來將持續深入探討。

Aging refers to a set of processes that lead over time to a gradual increase in vulnerability to be damaged and the probability of death. Fish is a promising model animal for aging research. It is well known that ayu (Plecoglossus altivelis), a common freshwater fish of East Asia, die after spawning and have a life span of only 1 year. The determinants for such a short life span are still ambiguous, but are probably connected with spawning. In the present study, we attempt to identify potential biomarkers in ayu as an aging animal model using proteomics approaches. Healthy ayu was collected from a fish farm and was grouped by gender and three growth stages: before, during, and after spawning. After measuring the biometric parameters, liver protein samples were extracted and cleaned-up, then subjected to two-dimensional electrophoresis (2-DE) analysis in triplicate experiments. Gel images were analyzed by Prodigy SameSpots software, and protein spots significantly altered by at least two-fold among three spawning stages were selected for LC-ESI-Q-TOF MS/MS analysis. Among nine proteins identified, betaine-homocysteine S-methyltransferase 1, triosephosphateisomerase, and glyceraldehyde-3-phosphate dehydrogenase were up-regulated, while apolipoprotein AI, 94 kD glucose-regulated protein, 78 kD glucose-regulated protein, tumor necrosis factor receptor, aldehyde dehydrogenase, and phosphoethanolaminemethyltransferase were down-regulated. We found that these proteins were associated with lipid transport and metabolism, carbohydrate metabolism, and protein folding-related diseases. Relationships of these liver proteins with the rapid death of ayu after spawning are now under investigation, and they may serve as potential markers for aging research.

摘要 V
ABSTRACT VII
縮寫表 (ABBREVIATIONS) VIII
第一章 研究背景與目的 1
第一節 老化 1
一、 老化的定義 1
二、 老化的發生 1
三、 探討老化的重要性 2
四、 目前的老化模式動物 4
五、 以香魚為老化動物模式 4
第二節 香魚簡介 6
一、 香魚的命名 6
二、 香魚的習性與特徵 6
三、 香魚的生命史 8
四、 香魚生長時期與環境壓力的判定 9
第三節 肝臟 10
一、 香魚的肝臟 10
二、 肝臟的生理功能 10
三、 肝臟的老化 11
第四節 蛋白質體學 12
第二章 材料與方法 14
第一節 藥品試劑 14
一、 採樣所需試劑 14
二、 蛋白質萃取、沈澱及定量 14
三、 二維凝膠電泳 14
第二節 儀器設備 16
第三節 生物材料 17
第四節 實驗方法 18
一、 香魚肝臟組織蛋白質萃取 (Total Protein Extraction Kit) 18
二、 蛋白質濃度定量 (Bradford dye-binding method) 19
三、 蛋白質沈澱試劑套組 (2-D Cleanup Kit) 19
四、 二維凝膠電泳 (2-DE) 20
五、 膠片影像擷取與分析 21
六、 膠體內水解與質譜儀分析 22
七、 Total RNA Extraction 22
八、 逆轉錄聚合酶連鎖反應 23
九、 瓊脂膠體電泳 24
十、 引子設計分析 24
十一、 即時定量聚合酶連鎖反應 (RT-qPCR) 24
第三章 結果 26
第一節 香魚的生物特徵 26
一、 八甲魚場 26
二、 頭城養殖場 27
三、 不同時期之雌性香魚性腺觀察 27
四、 不同時期之雄性香魚性腺觀察 28
第二節 二維凝膠電泳圖譜及圖譜分析 28
第三節 蛋白質身分鑑定 29
一、 表現量向上調的可能節蛋白質 29
二、 表現量向下調節的可能蛋白質 30
第四節 引子設計與分析 31
第五節 即時定量聚合酶連鎖反應 31
第四章 討論 32
第一節 香魚的生物特徵 32
第二節 二維凝膠電泳圖譜及圖譜分析 32
第三節 隨香魚老化而有顯著變化的蛋白 33
一、 載脂蛋白AI (Apo A-I) 33
二、 分子伴護蛋白 (Molecular chaperone) 34
三、 甜菜鹼同半胱氨酸S-甲基轉移酶-1 (BHMT-1) 36
四、 甘油醛-3-磷酸脫氫酶 (G3PDH) 37
第五章 結論與未來展望 40

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