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研究生:許亞婷
研究生(外文):Ya-Ting Hsu
論文名稱:椴木培養牛樟芝子實體酒精萃取物對大白鼠中大腦動脈阻塞缺血性中風之神經保護作用
論文名稱(外文):Neuroprotective Effects of Basswood Culture Antrodia cinnamomea Alcohol Extracts on Middle Cerebral Artery Occlusion Induced Focal Cerebral Ischemic Stroke In Rats
指導教授:龔瑞林龔瑞林引用關係
指導教授(外文):Zwe-Ling Kong
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:85
中文關鍵詞:缺血性中風牛樟芝抗氧化抗發炎神經保護
外文關鍵詞:Ischemia strokeAntrodia cinnamomeaAnti-oxidativeAnti-inflammatoryNeuroprotective
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在美國,每年有 750,000 件中風新案例出現,並且排名位居前十大死因第三名。缺血型中風占中風發生率 70%,而缺血型中風主要是因為血液供應減少,無法運輸氧氣和養分,進而造成神經功能缺損。牛樟芝為台灣最著名之傳統偏方,但目前很少人將牛樟芝探討其神經保護機制,因此本研究目的在於評估牛樟芝酒精萃取物在體外試驗部分是否可防止 H2O2 及 CoCl2 誘導下之氧化壓力傷害,並防止體內試驗在缺血再灌注下誘導之腦損傷。結果顯示,牛樟芝酒精萃取物具有清除自由基之能力,並可抑制 PC12 類神經細胞及 C6 膠質瘤細胞受到 H2O2 及 CoCl2 誘導下之脂質過氧化及活性物質 ROS 之生成,進而增加細胞存活率。以牛樟芝酒精萃取物 (385、770、1540 mg/kg) 管餵 SD 大鼠三週後可改善中大腦動脈梗塞手術 (MCAO) 所引起之 Nrf2 及 HO-1 mRNA 表現,下調發炎因子 TNF-α、iNOS 和 COX-2,增加缺氧誘導因子 HIF-1α 表現,並恢復缺血再灌注下之抗氧化酵素 GPx、CAT及 SOD 活性。此外,在缺血再灌注後其脂質過氧化表現隨著牛樟芝酒精萃取物劑量的提升呈現劑量依賴性地減少。因此本研究猜測牛樟芝酒精萃取物可預防缺血性中風是藉由抗氧化及抗發炎機制達到有效的神經保護作用。
Stroke is a common disease, and it is estimated that 750,000 new strokes occur annually in the U.S. and also is the third leading cause of death. Ischemia strokes accounted for the majority of stroke cases (70%), it occurs when the blood supply is diminished resulting in either a transient ischemic attack or a more long lasting neurological deficit due to an infarction in the brain. Antrodia camphorata (A. camphorata) is well-known in Taiwan as a traditional medicine. However, little is known about its neuroprotective mechanism. The purpose of this study was to evaluate the ability of A. camphorata alcohol extracts (AC-AE) to protect against H2O2 and CoCl2-induced oxidative stress in vitro, and use ischemia/reperfusion-induced brain injury in vivo. AC-AE scavenged the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). It also decrease lipid peroxidation and reactive oxygen species (ROS) in C6 and PC12 cell with IC50 value about H2O2 and CoCl2 , and increase cell survival. AC-AE dose (385、770、1540 mg/kg per day, p.o., 4 week) ameliorated the increase mRNA expression of Nrf2 and HO-1, downregulated inflammatory cytokine mRNA expression (TNF-α), incresed hypoxia inducible factor (HIF-1α) levels, restored the decrement in the glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) activitiy of serum and brain tissue caused by ischemia/reperfusion (I/R) in SD rat. Furthermore, it also dose-dependently inhibited the formation of lipid peroxidative products after I/R. These results suggest that AC-AE exerts effective prevention ischemia steoke against oxidative stress and hypoxia by mediating anti-oxidative, anti-inflammatory and neuroprotective mechanism.
謝誌 I
摘要 II
Abstract III
目次 VI
圖表目次 VIII
名詞縮寫 IX
壹、 緒論 1
貳、 文獻回顧 2
一、 腦血管疾病概述 2
(一)、 腦血管疾病現況 2
(二)、 現今通用治療方法 2
(三)、 缺血性腦中風成因 2
(四)、 自由基對腦部之損傷 3
(五)、 自由基對腦缺血性中風之損害途徑 4
(六)、 Nrf2/ARE抗氧化防禦訊息傳導對於中風的神經保護作用 5
(七)、 缺血下 HIF-1α 對神經之保護作用 6
二、 牛樟芝之介紹 7
(一)、 牛樟芝簡介 7
1. 牛樟芝學名之命名與分類 7
2. 牛樟芝型態 8
3. 牛樟芝培養法 8
(二)、 牛樟芝成分與生理活性 8
(三)、 安全性及毒性研究 9
(四)、 抗氧化功能之相關研究 9
(五)、 抗發炎功能之相關研究 10
(六)、 抑制腫瘤生長研究 10
(七)、 護肝功能之相關研究 10
(八)、 提升免疫力及調節免疫功能之研究 10
(九)、 保護神經之研究 11
參、 整體實驗設計 12
一、 細胞模式及流程 12
二、 動物分組及流程 13
肆、 實驗材料及方法 14
一、 材料 14
(一)、 牛樟芝來源 14
(二)、 動物來源 14
(三)、 細胞來源 14
(四)、 實驗藥品 14
(五)、 儀器設備 16
二、 方法 17
(一)、 牛樟芝酒精萃取物抗氧化分析 17
1. DPPH自由基清除能力 17
2. 還原力測定 18
(二)、 細胞模式 18
1. 細胞株解凍 18
2. 細胞株培養 18
3. 以過氧化氫 (H2O2) 誘導細胞氧化損傷 19
4. 低氧模擬劑 (CoCl2) 模擬腦部缺氧環境 19
5. 細胞存活率試驗 (MTT assay) 19
6. 脂質過氧化 (MDA) 測定 20
7. 利用流式細胞儀分析細胞內ROS含量 20
(三)、 動物模式 21
1. 動物飼養分組與樣品處理 21
2. 實驗動物分組 21
3. 中大腦動脈缺血與再灌流 (ischemia-reperfusion) 模式 22
4. 動物犧牲、採血和腦部摘取 22
5. 腦切片梗塞面積 (TTC) 染色分析 23
6. 超氧化物歧化酶 (Superoxide dismutase, SOD) 活性測定 23
7. 過氧化氫分解酶 (Catalase, CAT) 活性測定 23
8. 麩胱苷肽過氧化酶 (Glutathion peroxidase, GPx) 活性測定 24
9. ELISA分析血漿中細胞激素含量 24
10. Immunoblot分析腦萃取液中HIF-1α 蛋白質表現 25
11. 利用RT-PCR分析iNOS, COX-2, Nrf2, HO-1基因表現 27
(四)、 統計方法 29
伍、 結果與討論 30
(一)、 牛樟芝酒精萃取物其抗氧化能力分析 30
1. DPPH 自由基清除能力 30
2. 氧化還原能力 30
(二)、 牛樟芝酒精萃取物在氧化及低氧損傷下對神經細胞保護作用 30
1. 牛樟芝酒精萃取物對於細胞存活率之影響 30
2. 牛樟芝酒精萃取物對H2O2 及 CoCl2 誘導氧化及低氧損傷下其細胞
存活率之影響 31
3. 牛樟芝酒精萃取物對 H2O2誘導氧化壓力下 ROS 生成之影響 31
4. 牛樟芝酒精萃取物對CoCl2 誘導低氧損傷下 ROS 生成之影響 32
5. 牛樟芝酒精萃取物對 H2O2 和 CoCl2 誘導氧化及低氧損傷下造成脂
質過氧化之影響 32
(三)、 牛樟芝酒精萃取物對缺血性中風大鼠之影響 33
1. 牛樟芝酒精萃取物對中風體積之影響 33
2. 牛樟芝酒精萃取物對促發炎因子 (TNF-α) 表現之影響 33
3. 牛樟芝酒精萃取物對內生性抗氧化酵素之影響 33
4. 牛樟芝酒精萃取物對缺血性中風大鼠脂質過氧化作用之影響 34
5. 牛樟芝酒精萃取物對發炎因子 (iNOS、COX-2) 基因表現之影響 34
6. 牛樟芝酒精萃取物對內生性抗氧化反應基因 (Nrf2、HO-1) 表現之影
響 35
7. 牛樟芝酒精萃取物對缺氧誘導因子 (HIF-1α) 蛋白表現之影響 35
陸、 結論 36
參考文獻 37
圖表 49
附錄 70


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