臺灣博碩士論文加值系統

中文摘要
氧化壓力可能造成神經退化性疾病的重要因素，而抗氧化物質也顯示具有減低神經細胞氧化壓力所造成的細胞損傷。蝦青素為屬於類胡蘿蔔素，富含於海洋蝦蟹生物中，蝦青素已有研究指出其具有抗腫瘤、抗氧化、增強免疫力及清除自由基等多方面功能。本研究利用C6神經星狀膠質細胞具有星狀細胞及神經元細胞之特性，以此作為探討蝦青素神經保護作用的評估模式，實驗內容及作用指標包括：以細胞存活率評估毒性、以螢光分析活性氧產生量、劊蛋白酶(caspase)活性、鈣離子濃度、DNA片段化、流式細胞儀觀察膜電位變化之細胞凋亡關聯路徑及以ELISA及免疫墨點法觀察胞外訊號調控激酶的蛋白表現，也以阿茲海默氏症之重要病理特徵乙型類澱粉胜肽(beta-amyloid, A□)為探討核心；結果證實：第一部分，探討於氧化壓力下，蝦青素於50 □M以上之處理濃度在過氧化氫所製造的氧化壓力環境對於C6細胞凋亡具有抑制作用：DNA片段化現象減緩、caspase活性下降兩倍以及膜電位下降41.5％；第二部份，對於S100B此神經細胞所分泌之神經滋養蛋白，於蝦青素處理後隨著濃度的上升，其分泌量也逐漸增加；再者，首先評估蝦青素抑制乙型類澱粉胜肽(A□)纖維絲聚集試驗中發現，於50 □M蝦青素處理濃度下隨時間增加抑制纖維絲形成的比例由26.17 ﹪提升至67.7 ﹪。更進一步探討A□□於細胞內所誘發之神經發炎 (neuroimmflamation)，隨蝦青素添加濃度增加至100 □M，具有抑制細胞一氧化氮 (NO) 的產生、胞內鈣離子濃度上升及 ERK磷酸化的抑制情形達50 ﹪。本研究證實蝦青素可能藉由抑制凋亡路徑的訊息傳遞及降低神經發炎因子及蛋白的表現，得知其具有對抗氧化壓力及類澱粉胜肽堆積毒性的神經保護作用，將有潛力作為開發預防神經退化甚至阿茲海默氏症之關鍵素材。
關鍵字：蝦青素、神經保護、神經退化、過氧化氫、乙型類澱粉胜肽

Abstract
Recently, oxidative stress has been implicated in the progression of Alzheimer’s disease and antioxidants may protect neural cells from this attack. Astaxanthin (AST) is a carotenoid found in marine animals and alga. Several previous studies have demonstrated that AST exhibits a wide variety of biological activities including antioxidant, antitumor effects. In this study prove 50 □M AST reduce DNA fragmentation caused by H2O2 through inhibition of about 50% of caspase activity and 41.5% of mitochondria transmembrane potential. Morover inhibition from 26.17% to 67.7% of beta-amyloid(A□) fibrils formation is with time dependent manner. Further 100 □M AST can inhibit neuroinflammation factor include NO release, Ca2+ influx and 50% of ERK phosphorylation.
In conclusion, AST may against H2O2-/A□- induced neurotoxicity through inhibiting apoptosis signalling pathway and reducing expression of neuroinflammation factor and protein. Furthmore, AST has potential on apply to prevent neurodegenerative disorders (EX: Alzheimer’s disease).


Keywords: Astaxanthin, Neuroprotection, Hydrogen peroxide, Amyloid-□, apoptosis, Neuroinflamation

目錄
謝辭 I
名詞縮寫 (ABBREVIATION) III
中文摘要 IV
ABSTRACT VI
表目錄 X
圖目錄 XI
附錄 XII
壹、緒論 1
貳、文獻回顧 3
一、氧化壓力與神經毒性 3
1.自由基與氧化壓力 3
2.氧化壓力產生的神經毒性 4
3.氧化壓力與細胞凋亡 6
4.細胞凋亡參與神經退化性疾病發生 6
二、阿茲海默氏症( ALZHEIMER DISEASE, AD) 7
1. 阿茲海默氏症： 7
2. A□在阿茲海默氏症所扮演的角色 7
3. Amyloid precursor protein ( APP )： 8
4. □-amyloid導致神經細胞死亡的毒性機制 8
5. □-amyloid所引起的神經發炎(neuroinflammation) 9
三、神經保護作用 10
1. 神經退化性疾病成因 10
2. 氧化壓力與神經退化 10
3. 神經保護 11
四、蝦青素（ASTAXANTHIN） 12
1. 蝦青素 12
2. 蝦青素之生理功能 13
參、整體實驗設計 17
肆、實驗材料與方法 18
一、細胞株 18
二、藥品、試劑與儀器 18
三、實驗方法 23
1.C6神經膠瘤細胞株 (C6 glioma cell)的培養 23
2.細胞存活率試驗(MTT assay) 24
3. ROS 24
4. DNA fragmentation test 24
5. Caspase 3，Caspase 8，Caspase 9酵素活性之測定 25
6. 粒線體膜電位ΔΨm 之測定 26
7. 評估astaxanthin 抑制beta-amyloid 聚集之試管試驗 26
8. 胞內鈣離子濃度變化分析 27
9. NO釋放測定 (NO release) 27
10. ELISA 分析S100B濃度測定 28
11. RT-PCR分析iNOS, COX-2基因表現 28
12. Immunoblot 分析ERK1/2蛋白質表現量 31
13. 統計方法 35
伍、結果與討論 36
第一部份 ASTAXANTHIN對於氧化損傷(H2O2)環境之保護作用 36
一、Astaxanthin 對C6細胞於氧化壓力下細胞存活之影響 36
二、Astaxanthin 對C6細胞以H2O2 誘發產生胞內ROS之影響 37
三、Astaxanthin對C6細胞以H2O2 誘發產生細胞DNA片段化之影響 38
四、Astaxanthin抑制C6細胞以H2O2 誘發產生細胞凋亡之機制探討 38
第二部份 ASTAXANTHIN對於阿茲海默氏症其重要病理特徵BETA-AMYLOID之毒性引發神經發炎之保護作用 39
一、Astaxanthin對beta-amyloid (A□) fibrils 形成的影響 39
二、Astaxanthin 對C6細胞於beta-amyloid (A□)存在下細胞存活之影響 40
三、Astaxanthin 對C6細胞以A□ 誘發產生胞內ROS之影響 41
四、Astaxanthin對於胞內鈣離子濃度的影響 41
五、Astaxanthin對於A□刺激後所造成的NO釋放之影響 42
六、Astaxanthin對於A□所誘導一氧化氮合成酶(iNOS)mRNA表現之影響 43
七、Astaxanthin對於A□所造成的ERK磷酸化之影響 44
陸、總結 45
柒、參考文獻 46
捌、圖表 56
玖、附錄 73

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