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系統識別號 U0026-0102201016085000
論文名稱(中文) MA-1-PS對離體神經元保護:治療窗口與抗神經興奮毒性,抗氧化及清除自由基能力之探討
論文名稱(英文) Neuronprotection of MA-1-PS in vitro:therapeutic window and anti-excitotoxicity, antioxidant as well as free radical-scavenging action
校院名稱 成功大學
系所名稱(中) 生物科技研究所
系所名稱(英) Institute of Biotechnology
學年度 98
學期 1
出版年 99
研究生(中文) 黃巧雯
研究生(英文) Chiao-Wen Huang
學號 l6696404
學位類別 碩士
語文別 中文
論文頁數 79頁
口試委員 指導教授-吳天賞
指導教授-李宜堅
口試委員-陳宗鷹
中文關鍵字 缺血性腦中風  缺氧-葡萄糖實驗  神經元細胞  海馬迴組織  神經保護 
英文關鍵字 Ischemic stroke  Oxygen-glucose deprivation (OGD)  Neuronal cell  Hippocampal tissue  Neuroprotection 
學科別分類
中文摘要 摘要
在全世界腦中風是導致死亡重要的原因。常因缺乏有效的及時治療,引發短暫性局部大腦缺血的狀況,是由於興奮毒素神經傳導物質的釋放、鈣離子內流入神經元、自由基的產生和脂質過氧化,可因而產生不可逆的細胞損傷及神經行為不良,最終將導致細胞的壞死或細胞程式死亡的情形。
在研究中為採用之研究藥物MA-1-PS,是為MA-1的衍生物,而MA-1由菲律賓樟樹(Cinnamomum philippinense)萃取而來,MA-1 本為高脂溶性的化合物,由過去的研究已證實脂溶性MA-1為一抗氧化物及自由基清道夫,且可抑制血小板聚集,易通過血腦障壁治療急性缺血性腦中風,並發現強而有效的神經保護作用。但是當病患中風昏迷時,無法口服藥物,於是將MA-1進行化學結構修飾而產生的MA-1-PS,為水溶性,同時也增加溶解度,以致更易應用與達到提供靜脈注射的目的。
為證實MA-1-PS是否對神經元有相同之保護效果,研究中利用初代神經元培養 (primary neural culture)及海馬迴組織培養,並且利用缺氧及葡萄糖的實驗模式,模擬體內組織局部缺血而造成腦部損害的實驗。另外,進一步探討藥物是否可經由抑制脂質過氧化與清除自由基來達到保護神經的作用。
英文摘要 Abstract
Cerebral stroke is a leading cause of death worldwide. Because of the lack of efficient therapeutic alternatives, induced to transient focal cerebral ischemia that occurs during cerebrovascular surgery may result in irreversible cell damage and neurobehavioral dysfunction due to the release of excitotoxic neurotransmitters, influx of calcium ions into neurons, free radical generation, lipid peroxidation, and protein degradation. They eventually lead to necrotic and/or apoptotic cell death.
MA-1-PS is a derivative of MA-1. MA-1, a natural compound isolated from Cinnamomum philippinense. The agent is highly lipophilic. In the past researches have been demonstrated MA-1 was a strong natural antioxidant and free radical scavenger, had the potential to cross the blood-brain barrier to the brain and had neuroprotective properties. But when a serious patient is in a stupor, can not to oral drug by himself, so to modify MA-1, the product is MA-1-PS, is water soluble, moreover improve the solubility, can be easy to supply intravenous injection for therapy.
To confirm MA-1-PS whether has the same efficiency of neuroprotection. In experiment model, we use primary neural culture and organotypic hippocampal slice cultures, combined with oxygen-glucose deprivation ( OGD ), to mimic ischemia closely the situation in vivo result in brain disease. Further, to demonstrated that MA-1-PS have neuroprotection of ability via inhibiting lipidperoxidation and scavenging free radical.
論文目次 目錄
摘要 I
Abstract II
目錄 III
表目錄 VII
圖目錄 VIII
第一章 緒論 1
1-1 腦血管疾病介紹 1
1-1-1 腦血管疾病概論 1
1-1-2 腦中風的介紹 2
1-2 神經系統﹙Nervous system﹚ 3
1-2-1 中樞神經系統﹙Central nervous system,CNS﹚ 3
1-2-2 周邊神經系統﹙Peripheral nervous system,PNS﹚ 4
1-2-3 神經細胞﹙Nerve cell;Neuron﹚ 4
1-2-4 神經膠細胞﹙Glia cells﹚ 6
1-3 腦中風之流行病學 6
1-3-1 腦部之能量代謝與血流量的關係 6
1-3-2 大腦的血液循環﹙Cerebral circulation﹚ 7
1-3-3 缺血﹙Ischemia﹚/再灌流(Reperfusion)的傷害機制 9
1-4 解剖位置-海馬迴﹙Hippocampus﹚ 15
1-4-1 位置 15
1-4-2 功能 15
1-4-3 作用機轉 15
1-4-4 臨床醫學 17
1-5 實驗藥物 18
1-5-1 MA-1 18
1-5-2 MA-1-PS 19
1-6 缺氧及缺葡萄糖﹙Oxygen and glucose deprivation;OGD﹚ 20
1-7 研究計畫之目的 21
第二章 實驗材料與方法 22
2-1 實驗流程圖 22
2-2 實驗方法 25
2-2-1 初代神經元培養(Primary neural culture) 25
2-2-2 海馬迴組織培養 32
2-2-3 自由基清除試驗 38
2-2-4 抗氧化能力試驗 40
2-3 統計方法 41
第三章 結果 42
3-1 MA-1-PS對於體外培養神經元造成的細胞毒性 42
3-1-1 利用MTT方法測試MA-1-PS毒性 42
3-1-2 利用PI染色方法與量化證明MA-1-PS毒性 42
3-2 MA-1-PS抑制glutamate造成體外培養神經元的cell swelling 42
3-3 MA-1-PS抑制glutamate引發體外培養神經元的損傷 43
3-3-1 利用PI染色與量化實驗證明MA-1-PS可抑制glutamate造成神經元
的死亡 43
3-3-2 利用PI染色與量化實驗證明MA-1-PS在神經元細胞於glutamate後
不同時間點加入的保護效用 43
3-4 MA-1-PS可保護缺氧缺糖(OGD)造成體外培養神經元的損傷 43
3-4-1 利用PI染色與量化實驗證明MA-1-PS於缺氧缺糖實驗前的加入,可
抑制OGD造成神經元的死亡 44
3-4-2 利用PI染色與量化實驗證明MA-1-PS於缺氧缺糖實驗後不同時間點
加入的保護效用 44
3-5 MA-1-PS可保護缺氧缺糖(OGD)造成體外培養海馬迴的損傷 44
3-5-1 利用PI染色與量化實驗證明MA-1-PS於缺氧缺糖實驗前的加入,可
抑制OGD造成海馬迴組織的死亡 44
3-5-2 利用PI染色與量化實驗證明MA-1-PS於缺氧缺糖實驗後不同時間點
加入的保護效用 45
3-6 MA-1-PS清除自由基的能力 46
3-6-1 MA-1-PS之ABTS自由基清除試驗 ( ABTS radical
scavenging assay ) 46
3-6-2 DPPH 自由基清除試驗 ( DPPH radical scavenging assay ) 46
3-7 MA-1-PS抗氧化的能力 47
第四章 討論 48
4-1 MA-1-PS之溶解度與細胞毒性 48
4-2 MA-1-PS之神經保護效果 48
4-2-1 MA-1-PS可預防性地阻斷神經興奮性胺基酸 48
4-2-2 MA-1-PS可預防性地降低缺氧缺糖造成神經元細胞的損傷 49
4-2-3 MA-1-PS可預防性地降低缺氧缺糖造成海馬迴組織的損傷 50
4-3 MA-1-PS之修護時間有效性 50
4-3-1 MA-1-PS修護神經元細胞經神經興奮毒性損傷後之時間有效性 51
4-3-2 MA-1-PS修護神經元細胞經缺氧缺糖損傷後之時間有效性 51
4-3-3 MA-1-PS修護海馬迴組織經缺氧缺糖損傷後之時間有效性 51
4-4 MA-1-PS自由基清除之能力 52
4-5 MA-1-PS抗氧化之能力 52
4-6 MA-1-PS與MA-1之整合比較 52
第五章 結論 54
第六章 圖表 55
參考文獻 74

表目錄
表1-1 能量與血流量的關係 6
表3-1 MA-1-PS與α-Tocopherol、ascorbic acid以及MA-1 清除ABTS自
由基能力之比較 71
表3-2 MA-1-PS與α-Tocopherol、ascorbic acid以及MA-1 清除DPPH自
由基能力之比較 72
表3-3 MA-1-PS與α-Tocopherol、ascorbic acid以及MA-1 抗脂質過氧化
能力之比較 73

圖目錄
圖1-1 人體的神經系統 4
圖1 2 神經元結構示意圖 5
圖1-3 腦部主要的血管構成 8
圖1-4 腦部動脈組成之Willis氏動脈環 8
圖1-5腦部缺血產生神經毒素以致神經細胞死亡的機制 10
圖1-6腦缺血後各種生化物質的代謝變化 14
圖1-7 海馬迴解剖圖 15
圖1-8 海馬迴訊息傳遞途徑 16
圖1-9 顯微鏡下的海馬迴組織 17
圖1-10 菲律賓樟樹 18
圖1-11 MA-1的化學結構式 19
圖1-12 MA-1-PS 的化學結構式 20
圖2-1 實驗流程圖1 22
圖2-2 實驗流程圖2 23
圖2-3 實驗流程圖3 24
圖2-4 MTT化學反應式 28
圖2-5 生理切片機 34
圖2-6 ABTS自由基生成機制 39
圖2-7 DPPH自由基清除呈色機制 40
圖3-1 MTT測試MA-1-PS細胞毒性 55
圖3-2 PI染色法MA-1-PS細胞毒性 56
圖3-3 神經元細胞cell swelling 57
圖3-4 神經元細胞cell swelling統計圖 58
圖3-5 Pre-Glutamate神經元細胞PI染色 59
圖3-6 Pre-Glutamate神經元細胞PI染色之量化 60
圖3-7 Post-Glutamate神經元細胞PI染色 61
圖3-8 Post-Glutamate神經元細胞PI染色之量化 62
圖3-9 Pre-OGD神經元細胞PI染色 63
圖3-10 Pre-OGD神經元細胞PI染色之量化 64
圖3-11 Post-OGD神經元細胞PI染色 65
圖3-12 Post-OGD神經元細胞PI染色之量化 66
圖3-13 Pre-OGD海馬迴組織PI染色 67
圖3-14-1 DG區域中,各組死亡率比較 68
圖3-14-2 CA3區域中,各組死亡率比較 68
圖3-14-3 CA1區域中,各組死亡率比較 68
圖3-15 Post-OGD海馬迴組織PI染色 69
圖3-16-1 DG區域中,各組死亡率比較 70
圖3-16-2 CA3區域中,各組死亡率比較 70
圖3-16-3 CA1區域中,各組死亡率比較 70
圖3-17 ABTS 71
圖3-18 DPPH 72
圖3-19 抗脂質氧化能力 73

參考文獻 參考文獻
1.Kristian P. Doyle, Roger P. Simon, Mary P. Stenzel-Poore. Mechanisms of Ischemic Brain Damage . Review Article. Neuropharmacology.2008
2.Newman M.F, Grocott H.P, Mathew J.P. Report of the substudy assessing the impact of neurocognitive function on quality of life 5 years after cardiac surgery. Stroke. 2001;32:2874-2881
3.蔡宜殷. 以Melatonin 治療暫時性局部腦缺血白鼠有助其電生理及神經行為之改善.國立成功大學醫學工程研究所碩士論文.2003
4.http://stroke.tw/page/2-5-2.html
5.http://baike.baidu.com/view/9706.htm
6.http://life.nthu.edu.tw/~g864264/Neuroscience/neuron/cell.htm
7.http://content.edu.tw/junior/bio/tc_wc/textbook/ch05/text-ch05-2.htm
8.http://commons.wikimedia.org/wiki/File:Complete_neuron_cell_diagram_zh-tw.svg
9.朱復禮 (民81)‧臨床神經醫學‧臺北:合記。
10.吳進安 (民81)‧基礎神經學‧臺北:合記。
11.Heart Center Online (HCO);http://heart.healthcentersonline.com/stroke/tia.cfm
12.http://webanatomy.net/anatomy/circulatory_notes.htm
13.陳俐璇(民93)‧以鎂離子治療暫時性局部腦缺血白鼠之電生理及神經行為之研究‧國立成功大學醫學工程研究所碩士論文
14.Durukan A, Tatlisumak T. Acute ischemia stroke:Overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacology Biochemistry and Behavior 2007;87;179-197
15.Seok Joon Won, Doo Yeon Kim, Byoung Joo Gwag. Cellular and Molecular Pathways of Ischemic Neuronal Death. Journal of Biochemistry and Molecular Biology 2002;35;67-86
16.Siesjo B.K., Katsura K., Kristina T. Acidosis-related damage. Adv Neurol 1996;71;209-33;discussion 234-6
17.廖佩斐 (民96) ‧代謝性麩胺酸第五型作用調節劑NMDA 結抗劑對NMDA受體的抑制反應‧慈濟大學藥理暨毒理學研究所碩士論文
18.Dirnagl U., Iadecola C., Moskowitz M.A. Pathobiology of ischemic stroke:an integrate view. Trends Neurosci 1999;22;391-7
19.Takano K., Latour L.L., Farmato J.E., Carano R.A., Helamer K.G., Hasegawa Y., Sotak C.H., Fisher M. The role of spreading in focal ischemia evaluated by diffusion mapping. Ann Neurol 1996;39;308-18
20.Mergenthaler P., Dirnagl U., Meisel A. Pathophysiology of stroke:lessons from animal models. Metab Brain Dis 2004;19;151-67
21.Alexandra Loidl, Eva Sevcsik, et al. Oxidized phospholipids in mmLDL induce apoptotic signaling via activation of acid sphingomyelinase in arterial smooth muscle cells. J. Biol. Chem 2003;10;1074
22.Emerich D.F., Dean R.L., 3rd & Bartus R.T. The role of leukocyte following cerebral ischemia:pathogenic variable or bystander reaction to emerging infarct? Exp Neurol 2002;173;168-81
23.Love S. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol Psychiatry 2003;27;267-82
24.Blaine C. White, Jonathon M. Sullivan, Donald J. DeGracia, Brian J. O’Neil, Robert W. Neumar, Lawrence I. Grossman, Jose´ A. Rafols, Gary S. Krause. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. Journal of the Neurological Sciences 2000;179;1–33
25.http://www.psycheducation.org/emotion/hippocampus.htm
26.Lipski J, Wan CK, Bai JZ, Pi R, Li D, Donnelly D. Neuroprotective potential of ceftriaxone in in vitro models of stroke. Neuroscience 2007;146:617-629
27.http://scienceblogs.com/purepedantry/2007/04/stress_precedes_volume_reducti.php
28.http://www.bristol.ac.uk/synaptic/info/pathway/hippocampal.htm
29.Laura M.D, Crystal L.M, Dale R.C, Frederick C. Post-ischemia diazepam does not reduce hippocampal CA1 injury and does not improve hypothermic neuroprotection after forebrain ischemia in gerbils. Brain Research 2004;1013:223-229
30.George Hsiao, Che-Ming Teng, Joen-Rong Sheu, Yu-Wen Cheng, Kwok-Keung Lam, Yen-Mei Lee, Tian-Shung Wu, Mao-Hsiung Yen. Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger. Biochimica et Biophysica Acta 2001;1525;77-88
31.Sheu-Meei Yu, Tian-Shung Wu, Che-Ming Teng. Pharmacological characterization of cinnamophilin, a novel dual inhibitor of thromboxane synthase and thromboxane A2 receptor. Br. J. Pharmacol 1994;111;906-912
32.E.-Jian Lee, Hung-Yi Chena, Ming-Yang Lee, Tsung-Ying Chena, Yun-Shang Hsu, Yu-Ling Hu, Guan-Liang Chang, Tian-Shung Wu. Cinnamophilin reduces oxidative damage and protects against transient focal cerebral ischemia in mice. Free Radical Biology & Medicine 2005;39;495 – 510
33.余舒晴. MA-1對離體神經元保護:治療窗口與抗神經興奮毒性,抗氧化及清除自由基能力之探討.國立成功大學生物科技研究所碩士論文.2007
34.Pringle AK, Iannotti F, Wilde GJC, et al. Neuroprotection by both NMDA and non-NMDA receptor antagonists in in vitro ischemia. Brain Research 1997;755:36-46
35.Wang JP, Ho TF, Chang LC et al. J Pharm Pharmacol 1995;47:857-860
36.Anna R, Tobias C, Fredrik A, Sailasree N, Tadeusz W. Mouse Hippocampal Organotypic Tissue Cultures Exposed to In Vitro “Ischemia ”Show Selective and Delayed CA1 Damage That Is Aggravated by Glucose. J Cerebral Blood Flow &Metabolism 2003;23;(1):23-33
37.Kraus RL, Pasieczny R, Lariosa-Willingham K, Turner MS, Jiang A, TraugerJW. Antioxidant properties of minocycline: Neuroprotection in an oxidative stress Assay and direct radical-scavenging activity. J Neurochem 2005;94:819-827
38.Takayuki Negishi, Yoshiyuki Ishii, Shigeru Kyuwa, Yoichiro Kuroda, Yasuhiro Yoshikawa. Primary culture of cortical neurons, type-1 astrocytes, and microglial cells from cynomolgus monkey (Macaca fascicularis) fetuses. Journal of Neuroscience Methods 2003;131;133–140
39.G. Fotakis, J.A. Timbrell. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters 2006;160;171–177
40.http://en.wikipedia.org/wiki/File:Mttscheme.png
41.Gruol DL, Yu N, Parsons KL, Billaud JN, Elder JH, Phillips TR. Neurotoxic effects of feline immunodeficiency virus, FIV-PPR. J Neurovirol 1998;4;415-25
42.Matsumoto Y, Yamamoto S, Suzuki Y, Tsuboi T, Terakawa S, Ohashi N, Umemura K. Na+/H+ exchanger inhibitor, SM-20220, is protective against excitotoxicity in cultured cortical neurons. Stroke 2004;35;185-190
43.Johnson EM, Greenlund LJS, Akins PT, Hsu CY. Neuronal apoptosis:Current understanding of molecular mechanisms and potential role in ischemia brain injury. J.Neurotrama 1995;12;843-852
44.Lawrence EJ, Dentcheva E, Curtis KM, et al. Neuroprotection with delayed initiation of prolonged hypothermia after in vitro transient global ischemia. Resuscitation 2005;64;383-388
45.Lipski J, Wan CK, Bai JZ, Pi R, Li D, Donnelly D. Neuroprotective potential of ceftriaxone in in vitro models of stroke. Neuroscience 2007;146;617-629
46.Stoppini L, Parisi L, Oropesa C, Muller D. Sprouting and functional recovery in co-cultures between old and young hippocampal organotypic slices. Neuroscience 1997;80;1127-1136
47.Strasser U, Fischer G. Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation. J Neurosci Methods 1995;57;177-186
48.Laake JH, Haug FM, Wieloch T, Ottersen OP. A simple in vitro model of ischemic based on hippocampal slice cultures and propidium iodide fluorescence. Brain Res Protocols 1999;4;173-184
49.Arai K, Nishiyama N, Matsuki N, Ikegaya Y. Neuroprotective effects of lipoxygenase inhibitors against ischemic injury in rat hippocampal slice cultures.Brain Res 2001;904;167-172
50.ROBERT E. CHILDS and WILLIAM G. BARDSLEY. The Steady-State Kinetics of Peroxidase with 2,2'-Azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) as Chromogen. Biochem. J. 1975;145;93-103
51.Yamaguchi T, Takamura H, Matoba T, Terao J. Hplc method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2-picrylhydrazyl. Biosci Biotechnol Biochem 1998;62;1201-1204
52.Mir C, Clotet J, Aledo R, Durany N, Argemi J, Lozano R, Cervos-Navarro J, Casals N. Cdp-choline prevents glutamate-mediated cell death in cerebellar granule neurons. J Mol Neurosci. 2003;20;53-60
53.Won SJ, Kim DY, Gwag BJ. Cellular and molecular pathways of ischemic neuronal death. J Biochem Mol Biol. 2002;35;67-86
54.Buck J, Derguini F, Levi E, Nakanishi K, Hammerling U. Intracellular signaling by 14-hydroxy-4,14-retro-retinol. Science. 1991;254;1654-1656
55.Mosbacher J, Schoepfer R, Monyer H, Burnashev N, Seeburg PH, Ruppersberg JP. A molecular determinant for submillisecond desensitization in glutamate receptors. Science 1994;266;1059-1062
56.Tamura, H., & Shibamoto T, "Antioxidantive activity measurement and 4-hydroxy nonenal.", J. Am. Oil Chem. Soc. 1991;68;941–943
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