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系統識別號 U0026-2808201015583500
論文名稱(中文) 褪黑激素在中樞神經神經細胞中因缺血所引起的內質網壓力於活體與離體實驗中之影響
論文名稱(英文) The effects of melatonin on the CNS ischemia-induced neuronal endoplasmic reticulum stress in vivo and in vitro
校院名稱 成功大學
系所名稱(中) 生命科學系碩博士班
系所名稱(英) Department of Life Sciences
學年度 98
學期 2
出版年 99
研究生(中文) 洪家揚
研究生(英文) Chia-Yang Hung
學號 l5695402
學位類別 碩士
語文別 中文
論文頁數 62頁
口試委員 指導教授-張素瓊
共同指導教授-李宜堅
口試委員-吳天賞
口試委員-陳鴻儀
中文關鍵字 褪黑激素  內質網壓力  缺血性中風  中大腦動脈栓塞手術  缺氧-缺糖  初代神經元培養 
英文關鍵字 melatonin  endoplasmic reticulum stress  cerebral ischemia  middle cerebral artery occlusion (MCAO)  oxygen-glucose deprivation  primary neuronal culture 
學科別分類
中文摘要 缺血性中風 (ischemia stroke)為世界人口死因的前幾名,在中風的病例當中有80% 以上為缺血性中風,中風之後會造成神經功能的缺失與傷害,但目前還尚未找出有效的治療方法,因此近年的研究都在探討如何有效治療大腦的缺血性中風,致力找出腦神經細胞死亡之機制,並期望開發出新的藥物,用以治療缺血性中風所造成的神經細胞死亡;先前的許多研究中已經證實,褪黑激素為一良好的自由基清除劑,並且是一種神經保護劑,能夠減少因缺血性中風所引起的神經細胞死亡。在缺血性中風的機制方面,目前已經證實在中風之後會細胞會產生內質網壓力 (endoplasmic reticulum stress),抑制細胞合成蛋白質,進而造成神經細胞的傷害與死亡;因此本論文將分為兩個部份探討其相關機制: 第一: 以大白鼠來進行中大腦動脈栓塞 (Middle cerebral artery occlusion,MCAO ) 手術造成暫時性缺血性中風 (transient ischemia stroke),並於手術後給予褪黑激素 (melatonin, 5 mg/kg);第二: 以初代神經元培養(primary cortical mixed neuronal culture)在缺氧-缺糖 (Oxygen-glucose deprivation, OGD) 之刺激下,引發內質網壓力,並在事前給予褪黑激素 (melatonin),以觀察褪黑激素是否能夠減少內質網壓力,進一步達到保護細胞的效果;實驗結果發現大鼠手術後給予褪黑激素治療,並且在缺氧-缺糖刺激前,給予初代神經細胞不同濃度之褪黑激素,皆能夠降低內質網壓力其中RNA dependent protein kinase-like ER kinase (PERK) 途徑 (PERK pathway) 相關蛋白之表現。因此,從實驗結果可以推測,褪黑激素能夠藉由降低內質網壓力,以減少因中風所引起之神經細胞損傷與死亡;可能是經由降低細胞內之自由基 (free radicals),並活化細胞內抗氧化途徑之相關蛋白,以達到保護神經細胞的作用。
英文摘要 Stroke is a major public health problem in the worldwide. Ischemia stroke accounts for approximately 80% of all strokes and leads to neurologic morbidity and mortality; however, efficient theraputical treatments remain unavailable. Many studies evaluated melatonin is well known to be a potent free radical scavenger and an antioxidant and can decreased neuronal cell death following transient cerebral ischemia. Several recent reports indicate inhibitions of protein synthesis were inducible by endoplasmic reticulum stress under ischemia and that caused neuronal cell death. In the beginning of the experiments, we used Sprague-Dawley rats following transient middle cerebral artery (MCA) occlusion surgery and treat melatonin after surgery. Secondary, we gave an oxygen -glucose deprivation (OGD) stress in primary neuronal culture model and pretreatment of melatonin in different dosages. A series of results indicated melatonin can decrease the expression of phospho-PERK pathway related proteins in the MCAO model. We also found pretreatment of melatonin can decrease the expression of phospho-PERK pathway related proteins in primary neuronal culture under OGD stress. The possible effects of melatonin may scavenge free radical and induce antioxidant responds to decrease endoplasmic reticulum stress that can protect neuronal cells.
論文目次 中文摘要…………………………………………………………………...Ⅰ
Abstract……………………………………………………………………Ⅱ
誌謝………………………………………………………………………...Ⅲ
目錄…………………………………………………………………….......Ⅴ
表目錄……………………………………………………………………...Ⅷ
圖目錄……………………………………………………………………...Ⅷ
符號縮寫………………………………………………………………...... XI
第一章 緒論………………………………………………………………...1
1-1 內質網的構造與功能……………………………………………….....3
1-2 內質網壓力的產生與途徑………………………………………….....3
1-3 內質網壓力與神經退化性疾病…………………………………….....5
1-4 腦中風-缺血性中風……………………………………………………6
1-5 實驗藥物-褪黑激素……………………………………………………8
1-6 研究動機與目的……………………………………………………….9
第二章 實驗材料與方法………………………………………………….11
2-1 實驗流程……………………………………………………………...12
2-2 材料…………………………………………………………………...13
2-3 實驗方法……………………………………………………………...18
2-3.1 初代神經元培養……………………………………………………18
2-3.2 神經元細胞以缺氧-缺糖之刺激…………………………………...18
2-3.3 動物準備……………………………………………………………..19
2-3.4 S.D大白鼠中大腦栓塞手術 (MCAO)…………………………....20
2-3.5 動物犧牲……………………………………………………………..20
2-3.5 蛋白質抽取…………………………………………………………..21
2-3.6 蛋白質濃度測定……………………………………………………..22
2-3.7 西方點墨法分析……………………………………………………..22
2-3.8 免疫螢光染色分析…………………………………………………..23
第三章 結果………………………………………………………………..25
3-1 褪黑激素能夠抑制缺血性中風所造成的內質網壓力……………….25
3-2 褪黑激素能夠抑制初代神經元在缺氧-缺糖(OGD)刺激下所產生的
內質網壓力…..……..……………………………………………….....28
第四章 討論………………………………………………………………...32
4-1 褪黑激素降低內質網壓力,以達到保護神經細胞的效果.………….32
4-2 褪黑激素降低內質網壓力,可能與清除自由基的機制相關………..32
4-3 褪黑激素保護神經細胞的機制……………………………………......33
4-4 缺血性中風模式中,不同區域產生內質網壓力…………………......33
4-5 內質網壓力對於不同細胞的影響…………………………………......34
第五章 結論與展望………………………………………………………...35
5-1 褪黑激素抑制內質網壓力…………………………………………......35
5-2 褪黑激素在中風模式分子機制中的角色…………………………......35
第六章 統計圖表…………………………………………………………...36
第七章 參考文獻…………………………………………………………...56
附錄………………………………………………………………………….60
自述……………………………………………………………………….....62
參考文獻 Back, T. (1998). "Pathophysiology of the ischemic penumbra--revision of a concept." Cell Mol Neurobiol 18(6): 621-638.

Bertolotti, A., Y. Zhang, et al. (2000). "Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response." Nat Cell Biol 2(6): 326-332.

Boyce, M. and J. Yuan (2006). "Cellular response to endoplasmic reticulum stress: a matter of life or death." Cell Death Differ 13(3): 363-373.

Buonocore, G. and F. Groenendaal (2007). "Anti-oxidant strategies." Semin Fetal Neonatal Med 12(4): 287-295.

Cervantes, M., G. Morali, et al. (2008). "Melatonin and ischemia-reperfusion injury of the brain." J Pineal Res 45(1): 1-7.

Chen, H. Y., T. Y. Chen, et al. (2006). "Melatonin decreases neurovascular oxidative/nitrosative damage and protects against early increases in the blood-brain barrier permeability after transient focal cerebral ischemia in mice." J Pineal Res 41(2): 175-182.

Chen, H. Y., Y. C. Hung, et al. (2009). "Melatonin improves presynaptic protein, SNAP-25, expression and dendritic spine density and enhances functional and electrophysiological recovery following transient focal cerebral ischemia in rats." J Pineal Res 47(3): 260-270.

Chen, T. Y., M. Y. Lee, et al. (2006). "Melatonin attenuates the postischemic increase in blood-brain barrier permeability and decreases hemorrhagic transformation of tissue-plasminogen activator therapy following ischemic stroke in mice." J Pineal Res 40(3): 242-250.

Cheung, R. T. (2003). "The utility of melatonin in reducing cerebral damage resulting from ischemia and reperfusion." J Pineal Res 34(3): 153-160.
Cullinan, S. B. and J. A. Diehl (2006). "Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway." Int J Biochem Cell Biol 38(3): 317-332.
Cullinan, S. B., D. Zhang, et al. (2003). "Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival." Mol Cell Biol 23(20): 7198-7209.

DeGracia, D. J. (2004). "Acute and persistent protein synthesis inhibition following cerebral reperfusion." J Neurosci Res 77(6): 771-776.

DeGracia, D. J., R. Kumar, et al. (2002). "Molecular pathways of protein synthesis inhibition during brain reperfusion: implications for neuronal survival or death." J Cereb Blood Flow Metab 22(2): 127-141.

Gotoh, T. and M. Mori (2006). "Nitric oxide and endoplasmic reticulum stress." Arterioscler Thromb Vasc Biol 26(7): 1439-1446.

Hung, Y. C., T. Y. Chen, et al. (2008). "Melatonin decreases matrix metalloproteinase-9 activation and expression and attenuates reperfusion-induced hemorrhage following transient focal cerebral ischemia in rats." J Pineal Res 45(4): 459-467.

Kimball, S. R., A. Abbas, et al. (2008). "Melatonin represses oxidative stress-induced activation of the MAP kinase and mTOR signaling pathways in H4IIE hepatoma cells through inhibition of Ras." J Pineal Res 44(4): 379-386.

Koh, P. O. (2008). "Melatonin attenuates the cerebral ischemic injury via the MEK/ERK/p90RSK/bad signaling cascade." J Vet Med Sci 70(11): 1219-1223.

Koh, P. O. (2008). "Melatonin regulates nitric oxide synthase expression in ischemic brain injury." J Vet Med Sci 70(7): 747-750.

Kraus, R. L., R. Pasieczny, et al. (2005). "Antioxidant properties of minocycline: neuroprotection in an oxidative stress assay and direct radical-scavenging activity." J Neurochem 94(3): 819-827.

Kudo, T., S. Kanemoto, et al. (2008). "A molecular chaperone inducer protects neurons from ER stress." Cell Death Differ 15(2): 364-375.

Lee, E. J., M. Y. Lee, et al. (2005). "Melatonin attenuates gray and white matter damage in a mouse model of transient focal cerebral ischemia." J Pineal Res 38(1): 42-52.

Lee, E. J., T. S. Wu, et al. (2004). "Delayed treatment with melatonin enhances electrophysiological recovery following transient focal cerebral ischemia in rats." J Pineal Res 36(1): 33-42.

Lee, M. Y., Y. H. Kuan, et al. (2007). "Intravenous administration of melatonin reduces the intracerebral cellular inflammatory response following transient focal cerebral ischemia in rats." J Pineal Res 42(3): 297-309.

Li, F., T. Hayashi, et al. (2005). "The protective effect of dantrolene on ischemic neuronal cell death is associated with reduced expression of endoplasmic reticulum stress markers." Brain Res 1048(1-2): 59-68.

Li, J., M. Ni, et al. (2008). "The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells." Cell Death Differ 15(9): 1460-1471.

Lindholm, D., H. Wootz, et al. (2006). "ER stress and neurodegenerative diseases." Cell Death Differ 13(3): 385-392.

Liu, L., D. R. Wise, et al. (2008). "Hypoxic reactive oxygen species regulate the integrated stress response and cell survival." J Biol Chem 283(45): 31153-31162.
and redox homeostasis and enhancing cellular survival.

Ma, Y. and L. M. Hendershot (2004). "ER chaperone functions during normal and stress conditions." J Chem Neuroanat 28(1-2): 51-65.

Mehta, S. L., N. Manhas, et al. (2007). "Molecular targets in cerebral ischemia for developing novel therapeutics." Brain Res Rev 54(1): 34-66.

Morimoto, N., Y. Oida, et al. (2007). "Involvement of endoplasmic reticulum stress after middle cerebral artery occlusion in mice." Neuroscience 147(4): 957-967.

Newman, M. F., H. P. Grocott, et al. (2001). "Report of the substudy assessing the impact of neurocognitive function on quality of life 5 years after cardiac surgery." Stroke 32(12): 2874-2881.
Nicholls, D. and D. Attwell (1990). "The release and uptake of excitatory amino acids." Trends Pharmacol Sci 11(11): 462-468.

Paschen, W. (2003). "Endoplasmic reticulum: a primary target in various acute disorders and degenerative diseases of the brain." Cell Calcium 34(4-5): 365-383.

Pei, Z., S. F. Pang, et al. (2003). "Administration of melatonin after onset of ischemia reduces the volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model." Stroke 34(3): 770-775.

Picq, M., M. Dubois, et al. (1989). "Flavonoid modulation of protein kinase C activation." Life Sci 44(21): 1563-1571.

Schroder, M. (2006). "The unfolded protein response." Mol Biotechnol 34(2): 279-290.

Schroder, M. (2008). "Endoplasmic reticulum stress responses." Cell Mol Life Sci 65(6): 862-894.

Soto, C. (2003). "Unfolding the role of protein misfolding in neurodegenerative diseases." Nat Rev Neurosci 4(1): 49-60.

Tan, D. X., L. C. Manchester, et al. (2007). "One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species?" J Pineal Res 42(1): 28-42.

Truettner, J. S., K. Hu, et al. (2009). "Subcellular stress response and induction of molecular chaperones and folding proteins after transient global ischemia in rats." Brain Res 1249: 9-18.

White, B. C., J. M. Sullivan, et al. (2000). "Brain ischemia and reperfusion: molecular mechanisms of neuronal injury." J Neurol Sci 179(S 1-2): 1-33.

Witte, O. W., H. J. Bidmon, et al. (2000). "Functional differentiation of multiple perilesional zones after focal cerebral ischemia." J Cereb Blood Flow Metab 20(8): 1149-1165.

Wu, J. and R. J. Kaufman (2006). "From acute ER stress to physiological roles of the Unfolded Protein Response." Cell Death Differ 13(3): 374-384.
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