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研究生:周立哲
研究生(外文):Li-Jer Chou
論文名稱:參與腦死之網狀腹外側核區分子訊號蛋白質體研究
論文名稱(外文):Proteomic investigation of rostral ventrolateral medulla, a neural substrate intimately related to brain death
指導教授:張榮賢陳慶鏗陳慶鏗引用關係張雅雯張雅雯引用關係
指導教授(外文):L.S. ChangSamuel, H.H. ChanAlice, Y.W. Chang
學位類別:博士
校院名稱:國立中山大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:112
中文關鍵詞:腦死延腦鼻端腹外側核區組織氧量蛋白質體學抗氧化蛋白質ATP量
外文關鍵詞:proteomicsantioxidant proteinsATP contentstissue oxygenrostral ventrolateral medullabrain death
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當個體心血管系統和呼吸系統功能不可逆喪失或是全腦功能 (包含腦幹) 停止時稱為死亡,目前許多國家都以腦死做為法律上死亡定義。許多人會把植物人與腦死病人混淆,植物人對於本身或是週遭環境是沒有任何意識的。腦死病人與植物人在腦部受到嚴重損傷後都會陷入重度昏迷,與腦死病人不同的是植物人仍保有維持生命相關功能,例如心跳、呼吸與血壓維持。
存在於動脈壓中的“生與死”訊號源自於延腦鼻端腹外側核區,且與腦死有著密切的關聯性。動物實驗模擬腦死的模式下,藉由觀察動脈壓中 vasomotor components 功率密度的增加與減少,可發現在實驗動物趨向死亡的過程中,延腦鼻端腹外側核區中確實存在著 “pro-life” 及 “pro-death” 兩種程序。目前已經有許多分子機制與蛋白質被證實參與在這兩種程序中。延腦鼻端腹外側核區是與腦死有著密切關係的核區,但是其蛋白質體是否具有特異性仍有待進一步的研究。
為了解決這個疑問,本論文使用與植物人有直接關聯的大腦皮質來與延腦鼻端腹外側核區進行比較。利用二維凝膠電泳、介質輔助雷射脫附游離飛行式質譜儀與胜肽質量指紋法,探討延腦鼻端腹外側核區與大腦皮質在蛋白質體上的差異。
比較延腦鼻端腹外側核區與大腦皮質利用銀染呈色的二維凝膠電泳膠體,蛋白質點分佈的情形有 85.9 ± 2.3 % 相似度。在延腦鼻端腹外側核區與大腦皮質二維凝膠電泳膠體中分別鑑定出 318 點與 290點的蛋白質,同時根據功能將蛋白質分類成:binding activity、chaperone、antioxidant、oxidoreductase、 ubiquitin-proteasome system、cell cycle、catalytic activity、glycolysis、 tricarboxylic acid cycle、electron transport chain、endocytosis and exocytosis、 structural molecular function、apoptosis、transport、differentiation and neurogenesis、protein biosynthesis、cell junction 與 others。同時我們發現抗氧化相關蛋白質,包含 peroxiredoxin-1 (Prx-1)、Prx-2、Prx-5 與 Prx-6、thioredoxin 以及mitochondrial manganese superoxide dismutase 在延腦鼻端腹外側核區比大腦皮質有著較高的 mRNA 和蛋白質表現量。另一方面與 ubiquitin-proteasome system 相關的蛋白質,包含proteasome subunit alpha type-1、ubiquitin、uniquitin-conjugating enzyme E2 N、ubiquitin carboxyl-terminal hydrolase isozyme L1 與 L3,兩個區域在mRNA 與蛋白質表現量則是相似的。此外組織氧量、代謝性能量 (ATP) 與 ATP synthase alpha 和 beta 的蛋白質表現量,也是延腦鼻端腹外側核區高於大腦皮質。
延腦鼻端腹外側核區中有著較高的組織氧量與 ATP synthase subunits 暗示有機會生成較多的ATP,同時也提供細胞保護機制來對抗發生腦死過程中 ATP 衰減的機會,延腦鼻端腹外側核區中較高的組織氧量與高代謝性能量的生成伴隨著面臨著更多的氧化壓力,而高量抗氧化蛋白質則能提供補救以對抗更多的氧化壓力。
綜合以上結果,延腦鼻端腹外側核區中高表現量的抗氧化蛋白質、組織血氧、代謝性能量以及 ATP synthase alpha and beta 可提供共同的保護機制來對抗腦死。
An individual who has sustained either irreversible cessation of circulatory and respiratory functions, or irreversible cessation of all functions of the entire brain, including the brain stem is dead. Brain death is currently the legal definition of death in many countries. Many people confuse brain death with vegetative states. Patients in a vegetative state are unaware of themselves or their environment. Both patients with brain death and those in a vegetative state are unconscious following severe brain injury. Unlike the brain death, vegetative patient’s vital vegetative functions, such as cardiac action, respiration, and maintenance of blood pressure are preserved.
The rostral ventrolateral medulla (RVLM) is the origin of a “life-and-death” signal identified from systemic arterial blood pressure spectrum and intimately related to brain death. Based on the animal models of brain death, the observations that the power density of the vasomotor components of SAP signals undergoes both augmentation and reduction during the progression towards death strongly suggest that both ‘‘pro-life’’ and ‘‘pro-death’’ programs are present in the RVLM. A number of those ‘‘pro-life’’ and ‘‘pro-death’’ programs in the RVLM has now been identified along with their cellular and molecular mechanisms. As the neural substrate that is intimately related to brain death, one unresolved question is whether the proteome expressed in RVLM is unique.
To address the issue, we used the cerebral cortex, which is defunct under persistent vegetative state for comparison. 2-DE electrophoresis, MALTI-TOF MS and peptide mass fingerprinting were used for investigation the proteomic difference between the rat RVLM and cerebral cortex.
Quantitative analysis on silver-stained 2-DE electrophoresis gels revealed highly comparable distribution patterns of these protein spots for both brain regions, with 85.9 ± 2.3 % of protein spots from RVLM matched those from cerebral cortex. According to the protein function, these proteins were classed into binding activity, chaperone, antioxidant, oxidoreductase, ubiquitin- proteasome system, cell cycle, catalytic activity, glycolysis, tricarboxylic acid cycle, electron transport chain, endocytosis and exocytosis, structural molecular function, apoptosis, transport, differentiation and neurogenesis, protein biosynthesis, cell junction, and others. We found that a group of antioxidant proteins, including members of the peroxiredoxin (Prx) family (Prx-1, Prx-2, Prx-5, and Prx-6), thioredoxin and mitochondrial manganese superoxide dismutase exhibited significantly higher protein and mRNA expression levels in RVLM when compared to cerebral cortex. Tissue oxygen, ATP contents and ATP synthase subunits alpha and beta in RVLM were also significantly elevated. On the other hand, protein and mRNA levels of members of the ubiquitin-proteasome system, including proteasome subunit alpha type-1, ubiquitin, uniquitin-conjugating enzyme E2 N, ubiquitin carboxyl-terminal hydrolase isozyme L1 and L3, were comparable in both brain regions.
The presence of higher levels of tissue oxygen and ATP synthase subunits in RVLM, leading to augmented ATP production, provides a cellular safeguard mechanism to reduce the possibility of irreversible reduction in intracellular ATP contents that precipitate brain death. By manifesting an augmented tissue oxygen and metabolic energy production, RVLM is more prone to oxidative stress.
We conclude that a significantly elevated level of antioxidant proteins and mRNA in RVLM is consistent with the exhibition of higher tissue oxygen tension and metabolic energy production in this neural substrate, which together constitute a safeguard mechanism against brain death.
目 錄
論文審定書 .......................................................................................................... i
致謝 …………………………………………………………………………………… ii
中文摘要 …………………………………………………………….…………….... iii
英文摘要 ……………………………………………………………………….……... v
第一章 緒論 ……………………………………………………………………..….. 1
一、 腦死 (brain death) …………………………………………...….… 1
二、 動脈壓頻譜分析 (power spectral analysis of systemic arterial pressure signals) …..………………………………………...….… 1
三、 動脈壓頻譜中之 vasomotor components (LF與 VLF) 與腦死的關聯性 ………………………………………….……………...…… 2
四、 延腦鼻端腹外側核區 (rostral ventrolateral medulla, RVLM) 與腦死的關聯性 ………………………………………………..……….. 3
五、 在腦死的過程延腦鼻端腹外側核區中存在著 “pro-life” 與 “pro-death” 程序 ………………………………………..…….…... 3
六、 植物人 (vegetative state) 不等於腦死 …………….…………… 4
七、 蛋白質體學 ……………………………………………..………….. 5
八、 二維凝膠電泳 (two-dimensional gel electrophoresis) ……….... 6
九、 質譜儀技術 ………………………………………………………… 7
十、 基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 MS) ……………………………………...…………… 8
十一、 胜肽質量指紋 …………………………………………...…........ 9
第二章 研究動機與目的 ………………………………………………….………. 10
第三章 實驗材料與方法 ………………………………………………….………. 12
一、 動物準備 ……………………………………………………..…… 12
二、 組織取樣 ………………………………………………………….. 12
三、 蛋白質萃取與處理 ……………………………………………….. 12
四、 二維凝膠電泳 …………………………………………………….. 13
五、 膠體內水解 (in-gel digestion) 與基質輔助雷射脫附游離飛行式質譜儀 …………………………………………………………….. 14
六、 西方墨點浸漬分析 (Western blot analysis) …………..……….. 14
七、 核糖核酸的萃取 ………………………………………………….. 15
八、 反轉錄聚合酶鏈式反應 ………………………………………….. 15
九、 即時定量聚合酶鏈式反應 (real-time PCR) ...………………….. 16
十、 三磷酸腺苷 (adenosine triphosphate, ATP) 濃度測量 .…. 16
十一、 組織氧張力的測量 ……………………………….................... 16
十二、 統計分析 ………………………………………….................... 17
第四章 實驗結果 ………………………………………………………………….. 18
一、 比較大白鼠延腦鼻端腹外側核區與大腦皮質的蛋白質體間的差異 .……...………………………………………………………..... 18
二、 在抗氧化相關的蛋白質表現量與 mRNA 表現量,延腦鼻端腹外側核區都表現出較高的含量 …………………………………….. 19
三、 與分解蛋白質相關蛋白質延腦鼻端腹外側核區與大腦皮質有著相等的表現量 ……………………………………………………….. 19
四、 延腦鼻端腹外側核區表現出較高的組織氧量與能量生成 …….. 20
第五章 討論 ……………………………………………………….………………. 21
一、 延腦鼻端腹外側核區是高代謝能量需求的區域 ……………….. 21
二、 延腦鼻端腹外側核區擁有高度的抗氧化防禦機制 …………….. 22
三、 蛋白質分解相關的蛋白質於延腦鼻端腹外側核區與大腦皮質均有相等的表現量 …………………………….................................. 24
四、 延腦鼻端腹外側核區與大腦皮質蛋白質體研究上的差異 …….. 25
五、 阿茲海默症 (Alzheimer’s disease) 與抗氧化蛋白質的關聯性 ………………………………………………………………….. 26
第六章 結論與未來展望 ………………………………………………..………… 28
參考文獻 …………..………………………………………………………………… 30
附表、附圖 …………………………………………………………………………... 49

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