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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：134 下載：4
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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
                                

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