孫璐西臺灣大學:食品科技研究所邱泓智Chiu, Hung-ChihHung-ChihChiu2010-05-112018-06-292010-05-112018-06-292009U0001-1108200912153800http://ntur.lib.ntu.edu.tw//handle/246246/182209阿茲海默症是常見於老年人的神經退化疾病，導致患者記憶喪失與腦部萎縮。該病症的主因是由於患者腦部呈現類澱粉蛋白β胜肽amyloid-beta peptide (Aβ) 堆積，造成氧化壓力上升、發炎反應、神經元的損傷，進而造成腦部功能退化。PC-12細胞是一種類似副交感神經細胞的細胞株，能夠表現神經細胞的部分生理特性，廣泛應用於阿茲海默症與神經細胞相關研究。本研究將PC-12 細胞培養於含有合成的Aβ之培養基中，以模擬阿茲海默症中Aβ造成神經細胞損傷的情形。利用數種具有抗氧化能力或免疫調節功效的天然物，包含gallic acid、tetrahydrocurcumin、sesamol、proanthocyanidin A2、arachidin-1、以及巴西洋菇(Agaricus blazei) 液態發酵凍乾物的水萃物及乙醇萃物，與Aβ同時處理PC-12細胞，探討樣品保護PC-12細胞免受Aβ損傷的能力。多次進行長片段Aβ (Aβ1-40) 之聚集都未能展現細胞毒性，因此改以可展現毒性的Aβ25-35片段以及過氧化氫進行對PC-12之氧化傷害。結果顯示，分化後的PC-12細胞對於樣品的毒性更為敏感，然而對於過氧化氫的毒性卻較不敏感。Proanthocyanidin A2與arachidin-1都具有良好的保護效果，能減少Aβ25-35對於未分化PC-12 (nPC-12) 造成之傷害；同時在過氧化氫誘導的氧化壓力下，也都展現良好的保護能力。巴西洋菇的水萃物及乙醇萃物對於Aβ25-35毒性並無明顯保護效果，然而能夠保護過氧化氫對未分化PC-12所造成之傷害。在分化的PC-12 (dPC-12) 細胞中，所有樣品都未能有效減少過氧化氫所造成的毒性。在所使用的純化合物中，gallic acid、tetrahydrocurcumin、proanthocyanidin A2、arachidin-1都展現優於正控制組trolox的DPPH清除能力，sesamol及α-tocopherol為其次，而巴西洋菇萃取物DPPH清除能力則最差。結果顯示，proanthocyanidin A2與arachidin-1能夠有效的保護Aβ25-35對未分化PC-12細胞造成的毒性，並且推測樣品能夠有效的降低氧化壓力，進而保護細胞。巴西洋菇的水萃物及乙醇萃物能夠有效保護未分化PC-12細胞免於H2O2的傷害，並推測保護效果並非由於該樣品之抗氧化能力。Alzheimer’s disease (AD) is the most common form of dementia caused by accumulation of amyloid-beta (Aβ) peptide in the brain, leading to elevated oxidative stress, inflammation, and neuronal loss in the brain. PC-12 cells, a neuronal-like cell line, were treated with Aβ to simulate the toxicity of Aβ towards neuron cells in AD. Several samples with high antioxidative activity including gallic acid, proanthocyanidin A2, arachidin-1, and sesamol, which is able to pass through the blood-brain-barrier (BBB), and samples with immunomodulating activities including tetrahydrocurcumin, Agaricus blazei, were used in Aβ treated PC-12 cells to evaluate the potential of these samples in the prevention of AD. Several methods for the preparation of toxic aggregates of Aβ1-40 fragment failed to exhibit significant toxicity towards PC-12 cells. The shorter active fragment Aβ25-35 was used instead, and H2O2 was also applied to mimic the elevated oxidative stress observed in neurons treated by Aβ. Result showed that differentiated PC-12 cells formed neurites and were more sensitive to toxicity of samples, but more resistance towards oxidative stress induced by H2O2. Both proanthocyanidin A2 and arachidin-1 exhibited protective effect in naïve PC-12 cells towards both Aβ25-35 and H2O2 damage. Ethanol and water extracts of A. blazei did not protect naïve PC-12 cells from Aβ25-35 toxicity, but was effective in protection of H2O2 damage. However, all the samples failed to protect differentiated PC-12 cells from H2O2 insult. Of the pure compound used, gallic acid, tetrahydrocurcumin, proanthocyanidin A2, arachidin-1 all exhibited stronger DPPH scavenging ability than trolox, followed by sesamol and α-tocopherol, with the extracts of A. blazei exhibiting poor scavenging abilities. The results suggest that proanthocyanidin A2, arachidin-1 exhibited protection against Aβ25-35 in naïve PC-12 cells, possibly by decreasing the elevated oxidative stress caused by Aβ25-35. Ethanol and water extracts of A. blazei were poor in DPPH scavenging ability yet protected naïve PC-12 cells from H2O2, implicating mechanism of actions other than direct antioxidative activity.List of abbreviations i文摘要 iibstract iiiist of Figures viist of Tables viii Introduction 1I Literature Review 2I) Alzheimer’s disease 2II) Amyloid-beta peptide 6III) Clearance of Aβ 12IV) Aβ oligomer theory 14V) Tau hyperphosphorylation 14VI) Risk factors of AD 17. Genetic factors involved in AD 17. Cholesterol and AD 18. Metal ions and AD 18. Inflammation and AD 19. Oxidative stress and AD 22VII) Therapeutical approaches in AD 26. AChEI ( Acetylcholinesterase inhibitors ) 26. NMDA receptor antagonist 26. Secretase inhibitors 26. Immunization 27. Non-steroidal anti-inflammatory drugs (NSAIDs) 28. Statins 28. Metal chelators 29. Others 29VIII) PC-12 cell model 31IX) Aβ induced apoptosis 34II Experimental materials 36I) Ginkgo biloba Extract 36II) Gallic acid 36III) Tetrahydrocurcumin 36IV) Proanthocyanidin A2 37V) Sesamol 37VI) Arachidin-1 38VII) Agaricus blazei Murill. 38V Objective 42 Experimental design 43I Materials and methods 44I) Experimental samples 44II) Chemicals and reagents 44III) Equipments and instruments 47IV) Cell culture equipments 48V) Cell working solutions 49VI) Experimental procedures 50. Sample preparation 50. Preparation of aggregated Aβ1-40 51. Preparation of aggregated Aβ25-35 54. Cell culture 54. Cell seeding 55. Differentiation of PC-12 cells 55. MTT reduction assay 56. Cytotoxicity assay 56. Protection effect of samples against H2O2 57. Protection effect of samples against Aβ 580. Thioflavin T assay 581. DPPH scavenging assay 592. Tricine-SDS-PAGE for Aβ1-40 603. Native-PAGE for Aβ1-40 62II Results and discussion 64I) Sample toxicity towards nPC-12 and dPC-12 cells 64II) Preparation of Aβ1-40 for inducing PC-12 damage 66III) H2O2 toxicity in PC-12 cell 73IV) Protective effects of samples against H2O2 toxicity in nPC-12 76V) Protective effects of samples against H2O2 toxicity in dPC-12 77VI) Protective effect of samples against Aβ25-35 toxictiy in nPC-12 81VII) DPPH scavenging ability of samples 84III Conclusion 86X Future Work 87 References 111I Appendix 126application/pdf1961141 bytesapplication/pdfen-US阿茲海默症類澱粉蛋白PC-12 細胞Amyloid-betaPC-12 cellsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182209/1/ntu-98-R96641003-1.pdf