Examination of the interactions between amyloid proteins and cell membrane

目前發現，人體內至少有20種不同的蛋白質，與致病性的澱粉沈澱(amyloid deposits)的纖維組成(fibrillar components)有關。而因蛋白質發生大小或形狀上的變化，產生聚集，進一步造成類澱粉症(amyloidosis)，也就是蛋白質構象病(protein conformational disease)；其中，類澱粉β蛋白質(β-amyloid, Aβ)為導致阿滋海默症(Alzheimer's Disease)之主因，亦為這些蛋白質中相當重要之一種。 文獻顯示，類澱粉β蛋白質-細胞膜間交互作用與類澱粉β蛋白質誘發之神經毒性高度相關。研究亦發現，此交互作用可能受靜電作用力或細胞膜流動性所影響。然而，確定主宰此類澱粉β蛋白質與細胞膜間交互作用之機制至今仍無定論。 本研究提出：類澱粉β蛋白質-細胞膜間交互作用受與細胞膜性質息息相關之偶極電位(dipole potential)所影響之假說。認為偶極電位之變化反映類澱粉β蛋白質與細胞膜間之結合(binding)情形，而更進一步影響到誘發之毒性機制。因此吾人利用電場敏感性染劑(potential-sensitive fluorescent dye)配合雙波長螢光比值檢測法(dual-wavelength ratiometric fluorescence method)，量測細胞膜偶極電位受不同實驗條件變化之影響；並進一步觀察細胞膜偶極電位之改變，以對其間之交互作用作深入之探討與了解。結果顯示，當聚集狀態下之類澱粉症蛋白質與微脂粒作用時，可使其偶極電位下降；除此之外，添加膽固醇對於類澱粉β蛋白質所引起偶極電位下降之現象更加明顯。而吾人相信，本研究之結果可提供對於阿滋海默症及其他類澱粉症之機制更多的了解Up to now, at least twenty different human proteins and peptides have been isolated as the fibrillar components of disease-associated amyloid deposits. These proteins undergo aberrant changes in structure and accumulate to form aggregated species which lead to the amyloidogenic diseases. β-amyloid peptide (Aβ), the principal protein of Alzheimer’s disease, is also one of them. Previous studies showed that the interaction between Aβ and cell membrane is highly correlated to Aβ-elicited neurotoxicity. Several lines of evidence showed that the interaction between phospholipid membrane and Aβ might be associated with electrostatic-mediated forces or membrane fluidity. However, the detailed mechanism of this interaction remains largely unknown. In this work, we proposed a hypothesis that there is a link between membrane dipole potential and Aβ-cell membrane interaction, which is considered to be the first step in the mechanism of Aβ-induced toxicity and wish to test this hypothesis. To pursue our goals, we employed a dual-wavelength ratiometric fluorescence method with aid of the potential-sensitive fluorescent dye. At the outset, we sought out the optimal experimental conditions of the aforementioned ratiometric method. Next, we investigated the effect of binding of amyloid proteins and phospholipid vesicle on membrane dipole potential. Our results indicated that a decrease in dipole potential was observed upon binding of aggregated amyloid proteins to phospholipid membrane. In addition, a more dramatic change in membrane dipole potential was detected in phospholipid vesicles with cholesterol. We believe that our outcome may contribute to a better understanding of the mechanism(s) associated with AD and other amyloid diseases.第一章 緒論 1 第二章 文獻回顧 3 2-1 阿滋海默症(Alzheimer’s disease, AD)介紹 3 2-1-1 類澱粉症(Amyloidosis) 3 2-1-2 失智(dementia)與阿滋海默症(Alzheimer’s disease, AD) 4 2-1-3 阿滋海默症的分類 7 2-1-4 阿滋海默症的主要成因假說 8 2-2 阿滋海默症與類澱粉β蛋白質(β-amyloid, Aβ) 10 2-2-1 類澱粉前驅蛋白(amyloid precursor protein) 10 2-2-2 類澱粉β蛋白質 14 2-3 類澱粉β蛋白質與細胞膜間之交互作用 26 2-3-1 自由基生成(free radical generation) 26 2-3-2 鈣離子濃度平衡(calcium homeostasis) 28 2-3-3 類澱粉β蛋白質對細胞膜流動性(membrane fluidity)的影響 30 2-3-4 離子通道生成(ion channels formation) 31 2-3-5 靜電作用力對類澱粉β蛋白質與細胞膜交互作用之影響 32 2-3-6 類澱粉β蛋白質與細胞膜交互作用總結 33 2-4 微脂粒(Liposome)介紹 33 2-4-1 微脂粒簡介 33 2-4-2 微脂粒的分類 35 2-4-3 微脂粒的製備方法 35 2-4-4 微脂粒的安定性 39 2-5 偶極電位(Dipole potential)介紹 42 2-5-1 細胞膜(cell membrane)簡介 42 2-5-2 膜電位(membrane potential)介紹 44 2-5-3 膜電位之測量方法 49 第三章 研究動機 52 第四章 實驗裝置、藥品與步驟 53 4-1 實驗裝置 53 4-2 藥品 54 4-3 實驗步驟 55 4-3-1 PBS (phosphate buffered saline)之製備 55 4-3-2 微脂粒之製備 56 4-3-3 類澱粉β蛋白質Aβ(25-35)之備製 56 4-3-4 類澱粉β蛋白質Aβ(1-40)之備製 57 4-3-5 確實形成聚集狀態之類澱粉β蛋白質Aβ(1-40)備製 57 4-3-6 溶菌酶(lysozyme)之備製 58 4-3-7 雙波長螢光比值檢測法(dual-wavelength ratiometric fluorescence method) 58 第五章 結果討論 65 5-1 雙波長螢光比值檢測法(dual-wavelength ratiometric fluorescence method) 66 5-2 靜置時間之影響 70 5-3 不同溶劑對R值的影響 80 5-4 不同微脂粒濃度對R值的影響 83 5-5 類澱粉β蛋白質片段Aβ(25-35) 對細胞膜偶極電位的影響 85 5-6 類澱粉β蛋白質片段Aβ(1-40)對細胞膜偶極電位的影響 89 5-7 溶菌酶(hen agg white lysozyme)對細胞膜偶極電位的影響 93 5-8 膽固醇(cholesterol)與各物種和細胞膜偶極電位的關係 96 5-9 聚集狀態(aggregation state)之測試 100 5-10 總結：各物種和細胞膜偶極電位的關係 112 第六章 結論與建議 119 第七章 參考文獻 12

Exploring the Effects of Naturally Occurring Molecules on the Protein Aggregation

目前至少有27種，與人類有關之致死疾病(deadly human diseases)與其致病蛋白質錯誤折疊有關；雖然此類蛋白質其序列與功能皆無相似處，但與其相關之類澱粉症卻有相似之臨床、病理及生化特徵，即在人體內各患病組織產生結構與特性均相似之類澱粉纖維沉積。此外，研究亦已證實，即使不與人體疾病有關，蛋白質亦可利用不同環境條件誘使其產生類澱粉纖維結構；由此可知，即使是利用與疾病無關之蛋白質作為研究目標，透過研究其纖維化過程之特性及行為，依然可有助於了解如何抑制類澱粉蛋白之纖維化。本研究所選用之抑制物質皆為天然小分子，部分為常見於人體大腦內之灰質(gray / grey matter)中之磷脂質，薑黃素則為廣為人知之食品添加物及染色物質；兩者之角色均較人工合成之高分子物質、胜肽或是其他結構物，更易於取得，亦較易為人體吸收。 本研究之實驗結果可知，無論是磷脂質(己二酰甘油磷酸膽鹼(di-C6-PC)與庚二酰甘油磷酸膽鹼(di-C7-PC))或是多酚物質(薑黃素(curcumin))皆可明顯減少及延緩類澱粉蛋白(牛胰島素與雞蛋白溶菌酶)之纖維結構生成。另外，吾人更進一步發現利用兩細胞株(大白鼠嗜鉻性神經瘤細胞與人類神經瘤母細胞)，發現此兩蛋白質所形成之類澱粉纖維結構會對其產生明顯之毒殺性；且添加短鏈磷脂質與多酚物質皆可明顯保護兩細胞免於類澱粉纖維結構之毒殺。 除此之外，吾人續以不同溫度處理薑黃素，並發現部分溫度處理可產生較高比例之具抑制能力之薑黃素結構。利用紫外光 / 可見光光譜、電灑式質譜與核磁共振分析後得知，此一具抑制能力之結構極可能為薑黃素之雙體結構。 吾人亦藉由圓二色光譜分析與熱誘導開展分析，發現添加高濃度(4 mM)之磷脂質，與低溫(10Abnormally folded proteins have been associated with at least twenty-seven debilitating and deadly human diseases. These diseases have distinct clinical, pathological, and biochemical characteristics, resulting in amyloid deposits with similar structures, and their corresponding precursor proteins have unrelated functions and share little sequence homology. Moreover, mounting evidence has suggested that the formation of amyloid fibrils is also possible for a variety of non-disease associated proteins. By taking advantage of this generic fibril-forming property, investigations of the amyloid fibrillation using disease-irrelevant proteins can thus aid in our understanding of possible inhibition of amyloid aggregation. The inhibitors that we used in this study include the predominant short-chain phospholipids that are present in the gray matter of the human brain and the polyphenol (e.g.: curcumin) that is widely used in food spicing and coloring. These naturally-occurring small molecules are accessible and easily absorbed by human body as comparison to the other synthesized polymers, peptides, and molecules. Our results showed that all the short-chain phospholipids (di-C6-PC and di-C7-PC) and polyphenol (curcumin) are able to attenuate and retard the fibril formation of amyloid-forming proteins, such as bovine insulin and hen egg-white lysozyme. Also, we found that the fibrillar species of bovine insulin and hen egg-white lysozyme formed at acidic pH (pH 2.0) and elevated temperature (5

Maternal interactional styles and topic maintenance in mandarin mother-child conversational interactions

本研究的目的為探究中文母子對話中，母親的互動風格與話題延續的關係。 研究問題為: (a)本研究中兩位母親的互動風格為何? (b)兩位孩童的話題延續表現有何不同? (c)母親的互動風格與話題延續有怎樣的互動關係? 研究語料為兩組親子互動的自然語料，各取三個小時，孩童的年紀約為三歲。研究結果顯示兩位母親的互動風格有所不同，分別屬於命令型和引誘型。 而小孩的在話題延續上表現則是相近，大多用回應母親問題的方式來延續話題並且能在不同的情境中提供新資訊。對兩組親子互動的探究顯示命令型的母親比較不會延續對話而經常改變話題，造成較短的話題片斷。引誘型的母親則較注重對話的延續且較少改變話題，造成較長的話題片斷。The purpose of the study is to investigate the interaction of the maternal interactional styles and topic maintenance in Mandarin mother-child conversational interaction. There are three research questions: (a) What are the mothers’ interactional styles in the current case? (b) What are the differences among children’s competences of topic maintenance? (c) How do the maternal interaction styles interact with topic maintenance in the conversational interactions? Naturally occurring conversations of two dyads' conversational interactions were adopted as the data and analyzed. Results suggested that while the two mothers differed in terms of their interactional styles, the two children showed nearly equal sophistication in terms of their topic-maintaining competences. Examinations of the two dyads' interactions also revealed that the directive mother was less interested in maintaining the conversations, changed topics more frequently, and maintained discourse topics for less turns. The conversation-eliciting mother paid more attention on maintaining of the conversations, change topics less frequently, and maintained discourse topics for much more turns