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研究生: 黃景怡
Huang, Chimg-Yi
論文名稱: 利用初級細胞培養以及小鼠模式確認對阿茲海默氏症具治療潛力之中草藥
Identification of potential Chinese herbs for Alzheimer’s disease using primary culture and mouse models
指導教授: 謝秀梅
Hsieh, Hsiu-Mei
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: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 101
中文關鍵詞: NH037神經保護寡聚體Aβ認知焦慮
英文關鍵詞: NH037, neuroprotection, oligomeric Aβ, cognition, anxiety
DOI URL: https://doi.org/10.6345/NTNU202204367
論文種類: 學術論文
相關次數: 點閱:118下載:4
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  • 阿茲海默氏症(Alzheimer’s disease; AD)是目前最常見的漸進式神經退化性疾病,主要因海馬迴的神經元受損而導致認知功能障礙。據估計,全球有超過3500萬人罹患AD。AD起始的主要關鍵為類澱粉胜肽(beta-amyloid peptides; Aβ)堆積和tau蛋白質的過度磷酸化。近期有愈來越多的證據顯示,對於多因性之疾病AD而言,使用多目標特性的傳統中草藥治療效果可能優於單靶藥物。因此,近年來中草藥於AD的研究和治療越來越受到重視。在此研究中,我們建立了以寡聚體Aβ25-35和Aβ42處理的初級海馬迴神經細胞平台進行中草藥的篩選,希望藉此篩選出具有神經保護功效的中草藥。從此篩藥平台中,我們發現NH037中草藥能有效的增加神經元數目、神經突起長度、神經突起分支數目,並伴隨提高無活性GSK3β的量及減少Aβ堆積與脂質氧化壓力等神經保護效果。更進一步,於兩側海馬迴CA1區域注射寡聚體Aβ25-35的C57BL/6 小鼠動物實驗中,我們也發現NH037中草藥可以減少小鼠的焦慮及認知功能異常的問題,而在病理分析中我們可以看到NH037前處理減少Aβ類澱粉蛋白質堆積、tau蛋白質磷酸化、神經發炎反應與增加突觸相關蛋白質表現量及正腎上腺素神經元與血清素神經元數目以對抗寡聚體Aβ25-35所造成的細胞毒性。因此, NH037可能有潛力避免認知、非認知損傷以及誘發AD相關的病因特性。

    Alzheimer’s disease (AD) is the most common neurodegenerative disease associated with progressive damage in hippocampal neurons and cognitive dysfunctions. It is estimated that over 35 million people worldwide suffered from the disease. Both the accumulation of beta-amyloid peptides (Aβ) and tau protein phosphorylation are regarded as crucial events in the initiation of AD. Recently, more evidences show that the multi-target characteristics of traditional Chinese Herb Medicine (CHM) may be advantageous over single-target drugs against the multifactorial nature of AD. These drugs have therefore attracted much attention in the research and treatment in AD. In the present study, we established mouse primary hippocampal neuronal culture treated with oligomeric Aβ25-35 and Aβ42 as the screening platform of CHM. From the in vitro screening results , we found that CHM NH037 can prevent the decrease of neuronal number, neuritic length, branch number, lipid oxidation, amyloid deposition , and NH037 increasing the level of inactived GSK3β under neurotoxicity of oligomeric Aβ. Furthermore, the administration of NH037 also reduced anxiety and the cognitive impairment in the C57BL/6J mice treated with bilateral intrahippocampal CA1 injection of oligomeric Aβ25-35. From pathological analysis, we further found that the pretreatment of NH037 decreased the levels of Aβ deposition, tau protein phosphorylation, neuroinflammation, and increased the levels of synapse-related protein expression, noradrenergic and serotonergic neurons against the toxicity of oligomer Aβ25-35. Therefore, using CHM NH037 is a potential therapeutic strategy to prevent the cognitive, noncognitive dysfunction, and related pathogenic characterizations of AD.

    目錄 1.英文摘要 5 2.中文摘要 7 3. 縮寫表 9 4. 前言 11 4.1阿茲海默氏症 (Alzheimer’s disease) 11 4.3初級海馬迴神經元培養 13 4.4類澱粉蛋白質(Aβ) 13 4.6神經發炎反應 15 4.7 動物模式的建立 16 4.8中草藥做為治療AD之選擇 16 4.9中草藥葛根 (NH037) 18 5. 研究材料及方法 19 5.1動物 19 5.2中草藥的製備 19 5.3小鼠海馬迴初級神經元培養及藥物處理 19 5.4 Aβ42寡聚體之製備 20 5.5 Aβ25-35寡聚體的製備 21 5.6細胞免疫螢光染色分析(Immumocytochemical, ICC) 21 5.7乳酸脫氫酶分析(LDH assay) 22 5.8動物實驗與藥物處理 22 5.9動物行為分析(Behavioral analysis) 23 5.10免疫組織化學染色(Immunohistochemistry) 25 5.11西方墨點轉漬法(Western blot) 27 5.12統計分析 (Statistical analysis) 28 6. 結果 29 6.1單獨給予不同中草藥不影響初級海馬迴細胞之培養 29 6.2 NH021與NH037中草藥可以有效對抗Aβ42寡聚體所造成的細胞損傷 29 6.3 NH014,NH021與NH037中草藥可以有效對抗Aβ25-35寡聚體所造成 的細胞損傷 31 6.4 NH037在Aβ42寡聚體體外平台對神經細胞保護的分子機制分析 32 6.5 NH037在Aβ25-35寡聚體體外平台對神經細胞保護的分子機制分析 33 6.6 NH037緩解Aβ25-35寡聚體所誘發的焦慮行為但不影響自主運動(locomotor activity) 34 6.7 NH037減緩Aβ25-35寡聚體所誘發的短期記憶能力受損 35 6.8 NH037緩解Aβ25-35寡聚體誘導的認知功能受損 35 6.9 NH037可以有效緩解Aβ25-35寡聚體所造成的神經細胞tau蛋白質過度磷酸化路徑的活化情形 36 6.10 NH037能降低Aβ的堆積 38 6.11 NH037能有效避免Aβ25-35寡聚體所誘導的發炎反應情形 39 6.12 NH037能緩解因Aβ25-35寡聚體所誘發的突觸相關蛋白下降的情形 40 6.13 NH037可以有效緩解海馬迴CA1注射Aβ25-35寡聚體所造成之正腎上腺素及血清素神經元之傷害 40 6.14 NH037能夠顯著提升CAMKII的表現量 41 6.15 NH037不影響神經滋養因子(BDNF)的表現量 41 7. 討論 42 8. 參考資料46 9. 圖表 54

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