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研究生:謝育萍
研究生(外文):Yu Ping Hsieh
論文名稱:非創傷性蜘蛛膜下腔出血發生後人類和大鼠體內之F4-neuroprostanes、F2-isoprostanes或脂溶性抗氧化物的分析
論文名稱(外文):Analysis of F4-neuroprostanes, F2-isoprostanes, or Lipid-soluble Antioxidants in Humans and Rats following Non-traumatic Subarachnoid Hemorrhage
指導教授:顏秀娟顏秀娟引用關係
指導教授(外文):H. C. Yen
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:149
中文關鍵詞:動脈瘤破裂所引發的蜘蛛膜下腔出血F4-neuroprostanesF2-isoprostanes脂質過氧化一氧化氮脂溶性抗氧化物
外文關鍵詞:Aneurysmal subarachnoid hemorrhageF4-neuroprostanesF2-isoprostanesLipid peroxidationNitric oxideLipid-soluble antioxidants
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動脈瘤破裂所引發的蜘蛛膜下腔出血(aneurysmal subarachnoid hemorrhage,aSAH)是人類非創傷性的出血性中風中主要的一種。病人在發生aSAH之後,可能會發生延遲性的缺血性神經損傷,使其預後變差。我們假設病人在發生aSAH之後,會因為血紅素的釋放及缺血再灌流損傷,而使得體內的氧化壓力上升。F2-isoprostanes (F2-IsoPs)和F4-neuroprostanes (F4-NPs)分別是源自花生四烯酸(arachidonic acid)和二十二碳六烯酸(docosahexaenoic acid)最專一的脂質過氧化標誌。我們之前發表的結果顯示aSAH病人其腦脊髓液中的F2-IsoPs含量比控制組高,且與較差的臨床表徵和預後有正相關性。在現在這個研究中,我們已經完整建立起台灣第一個也是唯一一個以氣相層析/負離子化學游離質譜儀分析F2-IsoPs和F4-NPs的平台。然後我們進一步偵測十二位控制組與十五位aSAH病人其腦脊髓液中F4-NPs的含量,來探討神經元的氧化損傷在aSAH所扮演的角色。我們收集病人術後每天的腦脊髓液檢體來做偵測,最多收到術後第十天。結果顯示aSAH病人其F4-NPs含量明顯高於控制組,且與嚴重的出血和較差的預後有正相關性。此外,預後為死亡的aSAH病人,其腦脊髓液中的F4-NPs含量和F2-IsoPs相比,有較大的變動幅度且含量異常之高,尤其是在術後的前幾天,此情況更為明顯。另一方面,我們也測試實驗SAH引發血管痙攣之大鼠體內的氧化壓力是否有上升。打入一次自體血的大鼠,其氧化壓力與控制組相比並沒有差異。不過打入兩次自體血的大鼠,其血漿中輔酶Q10的含量與控制組相比,就有顯著之差異。總括而言,我們的結果是第一個提供aSAH病人其腦神經元有受到氧化損傷的證據,且腦脊髓液中的F4-NPs可能是比F2-IsoPs去預測病人預後更好之標誌。打入兩次自體血而引發蜘蛛膜下腔出血之大鼠可能是研究血管痙攣與氧化損傷關係較合適之模式。
Aneurysmal subarachnoid hemorrhage (aSAH) caused by aneurysmal rupture is a major type of non-traumatic hemorrhagic stroke in humans. Following aSAH, patients may develope delayed ischemic neurological deficit leading to poor outcome. We hypothesized that oxidative stress would be increased following aSAH due to hemoglobin release and ischemic-reperfusion injury. F2-isoprostanes (F2-IsoPs) and F4-neuroprostanes (F4-NPs) are the most specific markers of lipid peroxidation derived from arachidonic acid and docosahexaenoic acid, respectively. Our previously published results showed that levels of F2-IsoPs in cerebrospinal fluid (CSF) of aSAH patients were higher than controls and positively correlated with poor clinical conditions or outcome. In the present study, we have established the first and only complete platform to analyze F2-IsoPs and F4-NPs by gas chromatography/negative-ion-chemical-ionization mass spectrometry in Taiwan. We then further investigated the role of neuronal oxidative damage in aSAH by detecting F4-NPs in CSF from 12 controls and 15 aSAH patients. CSF of patients were collected everyday after surgery for up to 10 days. The results showed that the levels of F4-NPs in aSAH patients were significantly higher than controls and positively correlated with severity of hemorrhage and poor outcome. Moreover, CSF F4-NPs levels in aSAH patients with death outcome had greater variations and unusual high values, especially at early time points, compared with F2-IsoPs. On the other hand, we also examined whether oxidative stress was increased in rats with vasospasm induced by experimental SAH. The oxidative stress was not different between sham-control and single-hemorrhage rats. However, coenzyme Q10 levels in plasma of double-hemorrhage models were significantly different from sham-control rats. In summary, our results provide the first evidence for oxidative damage to neurons and indicate that CSF F4-NPs could be a better marker than F2-IsoPs for predicting outcome in aSAH patients. The double-hemorrhage model to induce SAH in rats may be more appropriate to investigate the relationship between vasospasm and oxidative damage.
指導教授推薦書
口試委員會審定書
長庚大學授權書 iii
誌謝 iv
中文摘要 vi
英文摘要 viii
目錄 x
第一章 研究背景 1
1.1 氧化壓力與脂質過氧化 1
1.1.1 F2-isoprostanes 3
1.1.2 F4-neuroprostanes 7
1.1.3 脂溶性抗氧化物 8
1.1.4 一氧化氮與peroxynitrite 11
1.2 蜘蛛膜下腔出血 14
1.2.1 SAH的成因及臨床診斷方法 14
1.2.2 在aSAH發生後所引發的血管痙攣與延遲性的缺血性神經損
傷 16
1.2.3 Experimental SAH的大鼠模式及其與aSAH病人的差別 17
1.2.4 非創傷性SAH與氧化壓力的關係 18
第二章 實驗假設與目的 22
第三章 實驗方法與材料 27
3.1 aSAH病人檢體的收集 27
3.2 Experimental SAH之大鼠模式 28
3.3 評估GC/NICI-MS的偵測極限 29
3.4 評估分析檢體中F4-NPs的含量時,所用之內標準品 30
3.5 萃取腦脊髓品管液中的F2-IsoPs和F4-NPs,並用GC/NICI-MS偵測
之,以評估這整個實驗流程的準確度 31
3.6 檢體中F2-IsoPs和F4-NPs含量之分析 31
3.7 大鼠橋腦組織中與血漿中各種脂溶性抗氧化物質含量的偵測 37
3.8 Nitrate/nitrite總量之偵測 38
3.9 統計分析方法 39
第四章 實驗結果 40
4.1 GC/NICI-MS的偵測極限 40
4.2 以GC/NICI-MS分析腦脊髓液中F2-IsoPs方法之建立,及整個實驗
流程準確度的評估 40
4.3 以GC/NICI-MS分析腦脊髓液中F4-NPs方法之建立,及整個實驗
流程準確度的評估 41
4.3.1 以[2H4]-15-F2t-IsoP作為測量檢體中F4-NPs時用的內標準品 41
4.3.2 以純的DHA氧化產生F4-NPs的結果來確定檢體中F4-NPs
peak的定量範圍 43
4.3.3 分析檢體中F4-NPs含量的整個實驗流程準確度之評估 43
4.4 aSAH病人腦脊髓液中的F4-NPs含量有顯著地上升 45
4.5 脂溶性抗氧化物在血漿和橋腦組織中的差別 47
4.6 Experimental SAH大鼠的橋腦組織和體液中各式氧化壓力指標 47
4.6.1 打入一次自體血引發SAH的2天後,其氧化壓力沒有顯著
變化 47
4.6.2 打入一次自體血引發SAH的1天後,其氧化壓力沒有顯著
變化 48
4.6.3 打入兩次自體血引發SAH的7天後,血漿中的CoQ10含量
顯著下降 49
第五章 討論與結論 50
5.1 建立以GC/NICI-MS分析檢體中F2-IsoPs和F4-NPs含量的方法 50
5.2 腦脊髓液中F4-NPs的含量與aSAH相關性之研究 54
5.3 Experimental SAH大鼠其體內氧化壓力指標之研究 59
參考文獻 66
圖 86
表 100
附圖 117
附表 127
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