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研究生:楊宗憲
研究生(外文):Zong-Xian Yang
論文名稱:建立高濃度氧誘導小鼠肺損傷模式及評估氣霧化胞外超氧歧化酶對肺部的保護效果
論文名稱(外文):Establishment of Hyperoxia-induced Acute Lung Injury (ALI) Mouse Model and Assessment of Aerosolized EC-SOD to Protect Lung Injury
指導教授:陳全木陳全木引用關係
指導教授(外文):Chuan-Mu Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
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:98
中文關鍵詞:胞外超氧歧化酶胞外超氧歧化酶
外文關鍵詞:extracellular superoxide dismutaseEC-SODacute lung injuryALI
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生物體具有抗氧化防禦系統以預防組織受到氧化傷害,其中,超氧化物歧化酶 (SOD) 能將超氧陰離子轉換成過氧化氫(H2O2),因而在抗氧化防禦系統中扮演重要角色。胞外超氧化物歧化酶 (EC-SOD) 為超氧化物歧化酶家族的成員之一,其存在於組織的胞外基質,尤其是肺部器官。在臨床中,多使用支持性氧氣療法治療病患呼吸困難。但不幸地,當病患給予支持性氧氣療法,常會造成急性肺損傷 (ALI)。在本篇研究,我們建立高濃度氧誘導急性肺損傷小鼠動物模式並以此模式評估肺部受損時給予重組人類胞外超氧化物歧化酶 (rhEC-SOD)投藥的保護效果。小鼠持續6天暴露在80%高濃度氧誘導肺損傷的環境下,並每天投予8小時氣霧化重組人類胞外超氧化物歧化酶 (實驗組) 或PBS (對照組)。試驗結果顯示,在PBS半數致死時間 (LT 50)為144小時,其肺部乾濕比相較rhEC-SOD處理有增加趨勢。而rhEC-SOD組別在144小時則有較低的肺部乾濕比及較高的存活率。此外,小鼠在80%高濃度氧誘導後,可觀察到肺泡細胞壁有明顯增厚、發炎細胞滲入並失去正常的肺泡構型。因此,我們推論在抗氧化防禦系統中的還原氧物質 (ROS) 可能會造成DNA、蛋白質、脂質的損傷。因此,本試驗利用瓊脂膠體電泳分析DNA斷裂和DNA慧星試驗計算DNA受損情形。試驗結果顯示,高濃度氧誘導肺損傷會發生細胞凋亡。透過免疫組織化學染色亦顯示rhEC-SOD能進入肺部,並藉由給予人類胞外超氧化物歧化酶則能延緩發炎反應之發生。

There is an anti-oxidant system to prevent tissue damage by oxygen in organism. In this system, superoxide dismutase (SOD) plays an important role that converts superoxide radicals (O2-) to hydrogen peroxide (H2O2). Extracellular superoxide dismutase (EC-SOD) is one of the members in SOD family. It was found in the extracellular matrix of tissues, especially in lung. The oxygen therapy is a common clinical intervention for patients with respiratory distress. Unfortunately, when patients are provided with oxygen therapy, it always causes acute lung injury. In this study, we established the hyperoxia-induced acute lung injury (ALI) mouse model and assessed the effects of recombinant human EC-SOD (rhEC-SOD) treatment on lung injury protection. The mice were exposed in 80% oxygen to induce acute lung injury for six days with nebulized rhEC-SOD or PBS treatment for eight hours per day. The results showed that lethal time (LT50) was 144 hours and the wet-to-dry ratio of lung tissue increased in PBS treatment. However, the mice treated with rhEC-SOD had lower lung wet-to-dry ratio and higher survival rate in 144 hours than in PBS treatment. In addition, mice in PBS treatment showed thickening of alveolar wall, much more inflammatory cell infiltration and loss of alveoli structure. These results were speculated that was correlated with anti-oxidant system. In this system, reactive oxygen species (ROS) of superoxide anion might cause damage in DNA, proteins, and lipids. According to the DNA ladder analysis and comet assay, the results suggested that apoptosis happened in hyperoxia induced ALI. Moreover, immunohistochemistry results also showed that rhEC-SOD was absorpted into the lung parenchyma obviously and postponed the inflammation induced by hyperoxia.  

誌謝 i
中文摘要 iii
Abstract iv
目次 v
表次 vii
圖次 viii
第一章 緒言 1
第二章 文獻探討 4
2.1 超氧化物歧化酶之分類 4
2.1.1銅鋅型超氧化物歧化酶 (Cu/Zn SOD) 6
2.1.2 錳型超氧化物歧化酶 (Mn SOD) 6
2.1.3 胞外超氧化物歧化酶 (EC-SOD) 9
2.2 超氧化物歧化酶抗氧化的機制 12
2.3 急性肺損傷 (acute lung injury) 15
2.3.1 急性肺損傷的成因 16
2.3.2 急性肺損傷與氧化傷害 16
2.3.3 肺炎和肺纖維化 19
2.4 肺損傷動物模式 21
2.4.1 機械式通氣 (mechanical ventilation) 誘導肺損傷模式 21
2.4.2 內毒素誘導肺損傷模式 23
2.4.3 高濃度氧誘導肺損傷模式 26
2.4.4 Bleomycin誘導肺損傷模式 29
2.4.5 油酸 (oleic acid) 誘導肺損傷模式 30
2.4.6 酸吸入 (acid aspiration) 誘導肺損傷模式 33
第三章 材料與方法 36
3.1 試驗動物 36
3.2 人類胞外超氧化物歧化酶活性測定-WST 39
3.3 致死率與組織切片 39
3.4 支氣管肺泡灌洗液 (bronchoalveolar lavage fluid , BALF) & 總細胞計數 (Total cell count) 41
3.5 不同血球型態計數 42
3.6 彗星試驗 (comet assay) for tissue 44
3.7 Pulmonary edema (wet/dry ratio) 46
3.8 免疫組織化學染色法 (Immunohistochemistry, IHC) 47
3.9 組織之RNA萃取與cDNA之製備 48
3.10 即時定量聚合連鎖反應 (real-time PCR) 50
3.11 DNA斷裂分析 (DNA fragmentation) 52
第四章 結果 55
4.1 高濃度氧誘導小鼠肺損傷模式之建立 55
4.2 小鼠肺損傷外觀與病理組織切片 56
4.3 肺水腫分析 56
4.4 SOD3經氣霧化後活性之變化 60
4.5外源性SOD3表現於小鼠肺部位置之分析 60
4.6 SOD3減少氧化性DNA傷害之分析 62
4.7 肺泡灌洗液中總細胞數之分析 62
4.9 以半定量聚合連鎖反應(semi-quantitative-PCR)分析肺部 前發炎因子的變化 67
4.10即時定量聚合連鎖反應 (Real-time PCR)分析肺部前發炎 因子的變化 72
第五章 討論 74
第六章 結論 76
參考文獻 77


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