短肽—小分子二元调节剂对β-淀粉样多肽聚集及神经毒性的调控

发布时间：2018-03-07 02:08

本文选题：淀粉样多肽　切入点：调节剂　出处：《天津大学》2014年硕士论文　论文类型：学位论文

【摘要】：阿尔兹海默症（AD）是影响世界人口健康的重要疾病之一。β-淀粉样多肽（Aβ）的聚集及其引发的神经毒性与AD的致病机制密切相关。采用分子调节剂调控Aβ的聚集行为、抑制其神经毒性是控制和治疗AD的有效途径之一。将不同类型的调节剂相耦合，有望综合调节剂与Aβ的多种作用力和多重作用位点，进而产生协同作用，，提高调节剂干扰Aβ组装聚集的能力。本论文提出了“短肽小分子二元调节剂”的概念和设计思路，实现了二者的高效协同，有效抑制了Aβ的聚集及神经毒性。主要研究结果如下：将短肽调节剂KLVFF和多酚调节剂白藜芦醇（Res）相耦合，设计了KLVFF Res二元调节剂。KLVFF作为Aβ聚集的关键序列，可通过疏水等相互作用特异性结合在Aβ的相应区域，而Res可干扰Aβ相邻主链之间氢键的形成，故二者能协同抑制Aβ的聚集。但低浓度下二元调节剂的协同作用较弱，可能是Res成氢键能力不强的缘故。选用成氢键能力更强的多酚类调节剂表没食子儿茶素没食子酸酯（EGCG）代替Res，发现KLVFF EGCG二元调节剂能产生明显的协同效应，有效抑制Aβ的聚集。借助圆二色光谱（CD）和原子力显微技术（AFM）表征，提出了EGCG和KLVFF产生协同效应的机理：KLVFF可通过疏水作用等特异性结合在Aβ的关键区域，而EGCG的强成氢键能力使它与Aβ的主链、侧链以及结合在Aβ上的KLVFF形成氢键，二者协同抑制了Aβ相邻主链基于疏水作用和氢键作用的密实堆积，进而抑制纤维的生长。因此，KLVFF EGCG二元调节剂有效地降低了Aβ的神经毒性，在较低剂量下细胞存活率即可达94%。此外，围绕癌症研究中循环肿瘤细胞（CTC）的富集这一关键问题，针对乳腺癌的CTC靶点蛋白人表皮生长因子受体2（HER2）设计了18条多肽，发现多肽GG19与HER2阳性的乳腺癌细胞系SK-BR3细胞结合特异性接近于HER2抗体，且亲合力较好，是替代HER2抗体的理想选择。将抗体和GG19连接到磁性纳米颗粒表面，分别制备了抗体-磁性纳米颗粒复合体和多肽-磁性纳米颗粒复合体，发现后者可成功从PBS缓冲液和新鲜血液两种体系中富集到SK-BR3细胞，富集率分别达到81.8%和70.4%，接近于抗体-磁性纳米颗粒复合体的富集率。因此，GG19有望代替抗体形成基于多肽的CTC富集与检测技术，对发展不依赖于抗体的CTC富集与检测技术具有实际意义。
[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.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91;R96

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