本文選題：淀粉樣多肽　切入點：調節(jié)劑　出處：《天津大學》2014年碩士論文　論文類型：學位論文

【摘要】：阿爾茲海默癥（AD）是影響世界人口健康的重要疾病之一。β-淀粉樣多肽（Aβ）的聚集及其引發(fā)的神經毒性與AD的致病機制密切相關。采用分子調節(jié)劑調控Aβ的聚集行為、抑制其神經毒性是控制和治療AD的有效途徑之一。將不同類型的調節(jié)劑相耦合，有望綜合調節(jié)劑與Aβ的多種作用力和多重作用位點，進而產生協(xié)同作用，，提高調節(jié)劑干擾Aβ組裝聚集的能力。本論文提出了“短肽 小分子二元調節(jié)劑”的概念和設計思路，實現了二者的高效協(xié)同，有效抑制了Aβ的聚集及神經毒性。主要研究結果如下： 將短肽調節(jié)劑KLVFF和多酚調節(jié)劑白藜蘆醇（Res）相耦合，設計了KLVFF Res二元調節(jié)劑。KLVFF作為Aβ聚集的關鍵序列，可通過疏水等相互作用特異性結合在Aβ的相應區(qū)域，而Res可干擾Aβ相鄰主鏈之間氫鍵的形成，故二者能協(xié)同抑制Aβ的聚集。但低濃度下二元調節(jié)劑的協(xié)同作用較弱，可能是Res成氫鍵能力不強的緣故。 選用成氫鍵能力更強的多酚類調節(jié)劑表沒食子兒茶素沒食子酸酯（EGCG）代替Res，發(fā)現KLVFF EGCG二元調節(jié)劑能產生明顯的協(xié)同效應，有效抑制Aβ的聚集。借助圓二色光譜（CD）和原子力顯微技術（AFM）表征，提出了EGCG和KLVFF產生協(xié)同效應的機理：KLVFF可通過疏水作用等特異性結合在Aβ的關鍵區(qū)域，而EGCG的強成氫鍵能力使它與Aβ的主鏈、側鏈以及結合在Aβ上的KLVFF形成氫鍵，二者協(xié)同抑制了Aβ相鄰主鏈基于疏水作用和氫鍵作用的密實堆積，進而抑制纖維的生長。因此，KLVFF EGCG二元調節(jié)劑有效地降低了Aβ的神經毒性，在較低劑量下細胞存活率即可達94%。 此外，圍繞癌癥研究中循環(huán)腫瘤細胞（CTC）的富集這一關鍵問題，針對乳腺癌的CTC靶點蛋白人表皮生長因子受體2（HER2）設計了18條多肽，發(fā)現多肽GG19與HER2陽性的乳腺癌細胞系SK-BR3細胞結合特異性接近于HER2抗體，且親合力較好，是替代HER2抗體的理想選擇。將抗體和GG19連接到磁性納米顆粒表面，分別制備了抗體-磁性納米顆粒復合體和多肽-磁性納米顆粒復合體，發(fā)現后者可成功從PBS緩沖液和新鮮血液兩種體系中富集到SK-BR3細胞，富集率分別達到81.8%和70.4%，接近于抗體-磁性納米顆粒復合體的富集率。因此，GG19有望代替抗體形成基于多肽的CTC富集與檢測技術，對發(fā)展不依賴于抗體的CTC富集與檢測技術具有實際意義。
[Abstract]:Alzheimer's disease (AD) is one of the most important diseases affecting the health of the world's population. The agglomeration of 尾 -amyloid polypeptide A 尾 and its neurotoxicity are closely related to the pathogenesis of AD. Molecular modulators are used to regulate the aggregation of A 尾. Inhibition of neurotoxicity is one of the effective ways to control and treat AD. Coupling different types of regulators is expected to synthesize the multiple forces and multiple action sites of A 尾, and then produce synergistic effects. In this paper, the concept and design idea of "short peptide and small molecule binary modulator" are proposed, which can achieve the high efficiency cooperation between the two kinds of modulators, and improve the ability of modulators to interfere with the assembly and agglomeration of A 尾. The aggregation and neurotoxicity of A 尾 were effectively inhibited. The main results were as follows:. By coupling short peptide modulator KLVFF with polyphenol modulator resveratrol, a binary regulator, KLVFF Res, was designed as the key sequence of A 尾 aggregation, which could be specifically bound in the corresponding region of A 尾 by hydrophobic interaction. However, Res can interfere with the formation of hydrogen bonds between adjacent main chains of A 尾, so they can synergistically inhibit the aggregation of A 尾, but the synergistic effect of binary modifiers at low concentration is weak, which may be due to the weak hydrogen bonding ability of Res. In this paper, the polyphenolics regulator with stronger hydrogen bonding ability was used to replace Res. it was found that the binary regulator of KLVFF and EGCG could produce obvious synergistic effect. By means of circular dichroism spectroscopy (CD) and atomic force microscopy (AFM), the mechanism of synergistic effect between EGCG and KLVFF was proposed. The strong hydrogen bonding ability of EGCG makes it form hydrogen bond with A 尾 main chain, side chain and KLVFF bound to A 尾, which synergistically inhibits the dense accumulation of A 尾 adjacent main chain based on hydrophobic and hydrogen bond interaction. Therefore, the binary regulator of KLVFF EGCG can effectively reduce the neurotoxicity of A 尾, and the cell survival rate can reach 94% at lower doses. In addition, a total of 18 polypeptides were designed for human epidermal growth factor receptor (EGF) receptor 2hER2, a CTC target protein in breast cancer, focusing on the enrichment of circulating tumor cells (CTCs) in cancer research. It was found that the binding specificity of polypeptide GG19 with HER2 positive breast cancer cell line SK-BR3 cells was close to that of HER2 antibody, and its affinity was good. It was an ideal alternative to HER2 antibody. The antibody and GG19 were connected to the surface of magnetic nanoparticles. The antibody magnetic nanoparticles complex and polypeptide magnetic nanoparticles complex were prepared respectively. It was found that the latter could be successfully enriched into SK-BR3 cells from two systems: PBS buffer and fresh blood. The enrichment rates were 81.8% and 70.4, respectively, which were close to the enrichment rate of the antibody magnetic nanoparticles complex, so GG19 could replace the antibody forming polypeptide based CTC enrichment and detection technique. It is of practical significance for the development of antibody independent CTC enrichment and detection techniques.
【學位授予單位】：天津大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R91;R96

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本文編號：1577468