國立臺灣師範大學博碩士論文全文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	蔡志誠 Tsai, Chih-Chen
論文名稱：	中草藥山楂的有效成分抑制阿茲海默氏症新的生物標誌Protein-X增加Amyloid-β毒性之研究 An active principle of Chinese herbal medicine, Crataegus maximowiczii, inhibiting the toxic amyloid-β induced by Alzheimer’s disease new biomarker-Protein-X
指導教授：	林榮耀 Lin, Jung-Yaw
學位類別：	碩士 Master
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	50
中文關鍵詞：	中草藥、阿茲海默氏症、X蛋白、類澱粉蛋白β
英文關鍵詞：	Chinese herbal medicine, AD, Protein-X, Aβ
DOI URL：	https://doi.org/10.6345/NTNU202202972
論文種類：	學術論文
相關次數：	點閱：135 下載：4
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

阿茲海默症 (Alzheimer’s disease) 是一種常見的神經退化性疾病，它的病理特徵主要有神經細胞的變性，蛋白質斑塊堆疊和神經纖維的纏繞。有許多的證據指出造成阿茲海默症最主要的病因是β類澱粉蛋白 (Aβ) 的堆疊。在人類的大腦當中，類澱粉蛋白會從小分子的單體 (monomers) 慢慢堆疊到低聚物 (oligomers)、纖維化 (fibrils)、最後變成斑塊 (plaques)。從以前的文獻中有指出Aβ的低聚合物比起其他的蛋白質型態是最具有毒性的，因此本研究是希望能從中草藥當中找出可以有效減少Aβ斑塊堆疊的小分子物質(SM-X) 進而減少Aβ的低聚合物。
本實驗室，目前已經發現Protein-X在年老的阿茲海默症老鼠當中比起年輕的小鼠有更多的表現而且伴隨著更多類Aβ低聚合物的堆疊。由建立Protein-X stably transfected 的細胞株中，在細胞存活度和西方墨點法的實驗中，可以看出有持續表現蛋白X的細胞中被外加的Aβ刺激之後會產生更多Aβ的聚集進而導致細胞的存活度降低，而從中藥萃取出的SM-X不僅能夠降低蛋白X所刺激出的 Aβ，也藉由此現象來達到拯救細胞的效果;因此，SM-X是一種能夠成為治療阿茲海默症的潛力藥物。

Alzheimer’s disease (AD) is the most common neurodegeneration disease. The pathologic characteristics are the degeneration of neuron cells, presence of neuritic plaques, and neurofibrillary tangles. Accumulating evidence suggests that the Amyloid β (Aβ) oligomers are the causative role in AD. In the human AD brain, Aβ monomers are prone to aggregate to oligomers, fibrils and eventually the plaques. Recently, many reports point out that Aβ42 oligomers have the greatest toxicity more than other types of Aβ. Thus,this study is to identify the active principle from Chinese herbal medicine that could effectively decrease Aβ oligomers.
In our laboratory, Protein-X has been found to be over expressed in aged triple transgenic AD mice (3xTg-AD) [PS1(M146V), APP(K670M/N671L), and tau(P301L)], accompanying with the increasing formation of Aβ oligomers. In cell-based system, Protein-X induced more Aβ oligomers aggregation and cytotoxicity is higher than that without Protein-X induction, but SM-X decreased the toxicity in above system. In cell-based western blot analysis, Proetin-X induced more Aβ oligomers,but SM-X also reduced the Aβ oligomerization induced by Protein-X. Collectively, SM-X could be a potential therapeutic agent for the treatment of AD.

中文摘要    I
Abstract    II
Introduction    1
   Alzheimer’s disease    1
   Amyloid-β formation    1
   The toxicity of Aβ    2
   Chinese herbal medicine    3
   Triple transgenic AD mouse model    4
Materials and Methods    5
   Materials    5
   Cell culture    6
   MTT assay    6
1.    Preparation of Aβ42 for MTT assay:    7
   Western blotting:    7
1.    Preparation of cell lysates    7
2.    Quantification of protein concentration    7
3.    Preparation of sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE)    8
4.    Protein sample preparation    9
5.    Electrophoresis    9
6.    Transferring the protein    9
7.    Immunoblotting    10
8.    Preparation of Aβ42 for cell treatment:    10
   ThT assay    11
1.    Preparation of Aβ42 for ThT assay:    11
2.  Methods of ThT assay    11
   Determination of Aβ42 oligomerization in cell-free system by Western blot analysis    11
1.    Preparation of the cell-free protein sample    11
2.  Aβ42 oligomerization in cell-free system by Western blot analysis    12
   Animal model    12
1.    Novel object recognition task    13
2.    Spontaneous alternation behavior Y-maze test    14
3.    Morris water-maze test    14
4    Preparation of protein sample of cerebral tissues    15
   Statistics    16
Results    17
   Aggregation of Aβ42 was increased with age accompanying with Protein-X elevation in the cerebra of 3xTg-AD mice.    17
   The cytotoxicity of Aβ42 was induced more by Protein-X in stable transfected Protein-X SH-SH5Y cell, of which could be reduced by the small molecule, SM-X.    17
   SM-X decreased Aβ42 oligomerization induced by Protein-X in SH-SH5Y cells, as demonstrated by Western blot analysis.    19
   SM-X decreased the formation of Aβ42 oligomers induced by Protein-X in dot blot assay.    20
   SM-X reduced Aβ oligomerization induced by Protein-X in cell-free system.    20
   SM-X decreased the formation of Aβ42 fibrils induced by Protein-X as measured by ThT analysis.    20
   SM-X improved the deficits of learning and memory in 3xTg-AD transgenic mice.    21
   SM-X treatment improved the behavior on the novel object recognition task in 3xTg-AD transgenic mice.    22
   SM-X treatment reversed behavior on spontaneous alternation behavior Y-maze test in 3xTg-AD transgenic mice.    22
Discussion    24
References    27
Figures    33
Tables    47
Supplementary data    48
                                

McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D. & Stadlan, E. M. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 34, 939-944, (1984).
Hardy, J. A. & Higgins, G. A. Alzheimer's disease: the amyloid cascade hypothesis. Science 256, 184-185, (1992).
Viola, K. L. & Klein, W. L. Amyloid beta oligomers in Alzheimer's disease pathogenesis, treatment, and diagnosis. Acta Neuropathologica 129, 183-206, (2015).
Stefani, M. & Dobson, C. M. Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. Journal of Molecular Medicine (Berl) 81, 678-699, (2003).
Lesne, S. E., Sherman, M. A., Grant, M., Kuskowski, M., Schneider, J. A., Bennett, D. A. & Ashe, K. H. Brain amyloid-beta oligomers in ageing and Alzheimer's disease. Brain 136, 1383-1398, (2013).
Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S., Denis, P., Fan, W., Kha, H., Zhang, J., Gong, Y., Martin, L., Louis, J. C., Yan, Q., Richards, W. G., Citron, M. & Vassar, R. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nature Neuroscience 4, 231-232, (2001).
O'Brien, R. J. & Wong, P. C. Amyloid precursor protein processing and Alzheimer's disease. Annual Review of Neuroscience 34, 185-204, (2011).
Zou, C., Montagna, E., Shi, Y., Peters, F., Blazquez-Llorca, L., Shi, S., Filser, S., Dorostkar, M. M. & Herms, J. Intraneuronal APP and extracellular Abeta independently cause dendritic spine pathology in transgenic mouse models of Alzheimer's disease. Acta Neuropathologica 129, 909-920, (2015).
Takahashi, R. H., Almeida, C. G., Kearney, P. F., Yu, F., Lin, M. T., Milner, T. A. & Gouras, G. K. Oligomerization of Alzheimer's beta-amyloid within processes and synapses of cultured neurons and brain. Journal of Neuroscience 24, 3592-3599, (2004).
Sakono, M. & Zako, T. Amyloid oligomers: formation and toxicity of Abeta oligomers. Federation of European Biochemical Societies Journal 277, 1348-1358, (2010).
Choi, Y. J., Chae, S., Kim, J. H., Barald, K. F., Park, J. Y. & Lee, S. H. Neurotoxic amyloid beta oligomeric assemblies recreated in microfluidic platform with interstitial level of slow flow. Scientific Reports 3, 1921, (2013).
Korshavn, K. J., Bhunia, A., Lim, M. H. & Ramamoorthy, A. Amyloid-beta adopts a conserved, partially folded structure upon binding to zwitterionic lipid bilayers prior to amyloid formation. Chemical Communications 52, 882-885, (2016).
DiChiara, T., DiNunno, N., Clark, J., Bu, R. L., Cline, E. N., Rollins, M. G., Gong, Y., Brody, D. L., Sligar, S. G., Velasco, P. T., Viola, K. L. & Klein, W. L. Alzheimer's Toxic Amyloid Beta Oligomers: Unwelcome Visitors to the Na/K ATPase alpha3 Docking Station. Yale Journal of Biology and Medicine 90, 45-61, (2017).
Necula, M., Kayed, R., Milton, S. & Glabe, C. G. Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. Journal of Biological Chemistry 282, 10311-10324, (2007).
Demattos, R. B., Lu, J., Tang, Y., Racke, M. M., Delong, C. A., Tzaferis, J. A., Hole, J. T., Forster, B. M., McDonnell, P. C., Liu, F., Kinley, R. D., Jordan, W. H. & Hutton, M. L. A plaque-specific antibody clears existing beta-amyloid plaques in Alzheimer's disease mice. Neuron 76, 908-920, (2012).
Wang, B. S., Wang, H., Wei, Z. H., Song, Y. Y., Zhang, L. & Chen, H. Z. Efficacy and safety of natural acetylcholinesterase inhibitor huperzine A in the treatment of Alzheimer's disease: an updated meta-analysis. Journal of Neural Transmission (Vienna) 116, 457-465, (2009).
Ho, Y. S., So, K. F. & Chang, R. C. Anti-aging herbal medicine--how and why can they be used in aging-associated neurodegenerative diseases? Ageing Research Reviews 9, 354-362, (2010).
Ho, Y. S., So, K. F. & Chang, R. C. Drug discovery from Chinese medicine against neurodegeneration in Alzheimer's and vascular dementia. Chinese Medicine 6, 15, (2011).
Sun, Z. K., Yang, H. Q. & Chen, S. D. Traditional Chinese medicine: a promising candidate for the treatment of Alzheimer's disease. Translational Neurodegeneration 2, 6, (2013).
Liu, H., Wang, J. & Tabira, T. Juzen-Taiho-To, an Herbal Medicine, Promotes the Differentiation of Transplanted Bone Marrow Cells into Microglia in the Mouse Brain Injected with Fibrillar Amyloid β. The Tohoku Journal of Experimental Medicine 233, 113-122, (2014).
Mazzanti, G. & Di Giacomo, S. Curcumin and Resveratrol in the Management of Cognitive Disorders: What is the Clinical Evidence? Molecules 21, 1-27, (2016).
Luo, C., Ke, Y., Yuan, Y., Zhao, M., Wang, F., Zhang, Y. & Bu, S. A novel herbal treatment reduces depressive-like behaviors and increases brain-derived neurotrophic factor levels in the brain of type 2 diabetic rats. Neuropsychiatric Disease and Treatment 12, 3051-3059, (2016).
Elder, G. A., Gama Sosa, M. A. & De Gasperi, R. Transgenic mouse models of Alzheimer's disease. Mount Sinai Journal of Medicine 77, 69-81, (2010).
Stockert, J. C., Blazquez-Castro, A., Canete, M., Horobin, R. W. & Villanueva, A. MTT assay for cell viability: Intracellular localization of the formazan product is in lipid droplets. Acta histochemica 114, 785-796, (2012).
Marks, D. C., Belov, L., Davey, M. W., Davey, R. A. & Kidman, A. D. The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells. Leukemia Research 16, 1165-1173, (1992).
Gade Malmos, K., Blancas-Mejia, L. M., Weber, B., Buchner, J., Ramirez-Alvarado, M., Naiki, H. & Otzen, D. ThT 101: a primer on the use of thioflavin T to investigate amyloid formation. Amyloid 24, 1-16, (2017).
Lindberg, D. J., Wenger, A., Sundin, E., Wesen, E., Westerlund, F. & Esbjorner, E. K. Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction. Biochemistry 56, 2170-2174, (2017).
Antunes, M. & Biala, G. The novel object recognition memory: neurobiology, test procedure, and its modifications. Cognitive Processing 13, 93-110, (2012).
Kealy, J., Bennett, R., Woods, B. & Lowry, J. P. Real-time changes in hippocampal energy demands during a spatial working memory task. Behavioural Brain Research 326, 59-68, (2017).
Morris, R. Developments of a water-maze procedure for studying spatial learning in the rat. Journal of Neuroscience Methods 11, 47-60, (1984).
Oddo, S., Caccamo, A., Kitazawa, M., Tseng, B. P. & LaFerla, F. M. Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease. Neurobiology of Aging 24, 1063-1070, (2003).
Oddo, S., Caccamo, A., Tran, L., Lambert, M. P., Glabe, C. G., Klein, W. L. & LaFerla, F. M. Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of Alzheimer disease. A link between Abeta and tau pathology. Journal of Biological Chemistry 281, 1599-1604, (2006).
Podlisny, M. B., Ostaszewski, B. L., Squazzo, S. L., Koo, E. H., Rydell, R. E., Teplow, D. B. & Selkoe, D. J. Aggregation of secreted amyloid beta-protein into sodium dodecyl sulfate-stable oligomers in cell culture. Journal of Biological Chemistry 270, 9564-9570, (1995).
Walsh, D. M., Tseng, B. P., Rydel, R. E., Podlisny, M. B. & Selkoe, D. J. The oligomerization of amyloid beta-protein begins intracellularly in cells derived from human brain. Biochemistry 39, 10831-10839, (2000).
Townsend, M., Shankar, G. M., Mehta, T., Walsh, D. M. & Selkoe, D. J. Effects of secreted oligomers of amyloid beta-protein on hippocampal synaptic plasticity: a potent role for trimers. Journal of Physiology 572, 477-492, (2006).
Cleary, J. P., Walsh, D. M., Hofmeister, J. J., Shankar, G. M., Kuskowski, M. A., Selkoe, D. J. & Ashe, K. H. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nature Neuroscience 8, 79-84, (2005).
Funato, H., Enya, M., Yoshimura, M., Morishima-Kawashima, M. & Ihara, Y. Presence of sodium dodecyl sulfate-stable amyloid beta-protein dimers in the hippocampus CA1 not exhibiting neurofibrillary tangle formation. American Journal of Pathology 155, 23-28, (1999).
Roher, A. E., Chaney, M. O., Kuo, Y. M., Webster, S. D., Stine, W. B., Haverkamp, L. J., Woods, A. S., Cotter, R. J., Tuohy, J. M., Krafft, G. A., Bonnell, B. S. & Emmerling, M. R. Morphology and toxicity of Abeta-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease. Journal of Biological Chemistry 271, 20631-20635, (1996).
Kawarabayashi, T., Shoji, M., Younkin, L. H., Wen-Lang, L., Dickson, D. W., Murakami, T., Matsubara, E., Abe, K., Ashe, K. H. & Younkin, S. G. Dimeric amyloid beta protein rapidly accumulates in lipid rafts followed by apolipoprotein E and phosphorylated tau accumulation in the Tg2576 mouse model of Alzheimer's disease. Journal of Neuroscience 24, 3801-3809, (2004).
Oh, K. J., Perez, S. E., Lagalwar, S., Vana, L., Binder, L. & Mufson, E. J. Staging of Alzheimer's pathology in triple transgenic mice: a light and electron microscopic analysis. International Journal of Alzheimer’s disease 2010, 780102, (2010).
Attar, A., Liu, T., Chan, W. T., Hayes, J., Nejad, M., Lei, K. & Bitan, G. A shortened Barnes maze protocol reveals memory deficits at 4-months of age in the triple-transgenic mouse model of Alzheimer's disease. PLoS One 8, e80355, (2013).
Filali, M., Lalonde, R., Theriault, P., Julien, C., Calon, F. & Planel, E. Cognitive and non-cognitive behaviors in the triple transgenic mouse model of Alzheimer's disease expressing mutated APP, PS1, and Mapt (3xTg-AD). Behavioural Brain Research 234, 334-342, (2012).

簡易檢索 / 詳目顯示

相關論文