【作者】 宾艳南；

【导师】 向娟；

【作者基本信息】 中南大学 ， 化学， 2013， 博士

【摘要】 p-淀粉样多肽(amyloid β-peptide,Aβ)是阿尔茨海默病(Alzheimer’s disease, AD)病理学特征——淀粉样斑块的主要成分,与AD的发生密切相关。作为传导神经突触信号的重要介质,Cu2+、多巴胺(dopamine, DA)、去甲肾上腺素(norepinephrine, NE)等小分子可与Ap发生作用,对脑部神经元产生不同的影响。为了深入理解Ap与神经传导介质小分子的相互作用对AD病理发展的影响,本论文分别研究了Cu2+、DA、NE与Ap相互作用的机制,获得了相关动力学参数,并初步探讨了相关体系在AD病理中的作用。本论文主要工作包括：(1)获得Aβ-Cu2+络合物的解离常数是探讨AD治疗方案的基础之一,但现有文献报道中没有涉及到酸性条件下的Aβ-Cu2+解离常数。本论文采用表面等离子体激元共振(surface plasmon resonance,SPR)技术研究了酸性条件下Ap与Cu2+相互作用的动力学,发现随着溶液环境从中性(pH7.0)到弱酸性(pH4.0), Aβ-Cu2+的解离常数从3.5×10-8mol·L-1增大到8.7×10-3mol·L-1。通过定量检测-OH的产生量,发现酸性环境下Cu2+催化产生·OH的能力增强,毒性增大；随着pH的升高,Aβ结合Cu2+的能力增强,抑制了Cu2+催化产生·OH的反应,降低了其细胞毒性。(2)溶剂的选择影响Ap-Cu2+解离常数的检测。通常认为Tris是检测Aβ-Cu2+解离常数的理想溶剂。但作为亲核试剂,Tris对Aβ/Cu2+相互作用的影响尚不清楚。本论文采用多种方法系统研究了不同环境条件下Tris对Aβ/Cu2+相互作用的影响。透射电镜和电喷雾电离质谱结果证明酸性条件下可形成Tris-Aβ-Cu2+三元络合物。采用SPR、紫外吸收光谱和电化学技术发现,Tris对Aβ/Cu2+相互作用的影响：Kd增大一个数量级；阻碍Ap与Cu2+之间的电荷迁移,减弱其相应的紫外吸收；Aβ-Cu2+的氧化电位正移。通过定量检测-OH的产生量发现,由于Tris对于Aβ络合Cu2+的反应有竞争作用,使Aβ1-42-Cu2+的解离常数增大,溶液中游离Cu2+增多,所以Tris溶液中-OH的产量增大。中性条件下Tris与Ap无相互作用,不利于三元络合物的生成。(3)研究Ap与DA的相互作用机理可解释DA在Ap的毒性和AD的发展中所起的作用。采用SPR和电化学技术研究了亲水段Aβ1-16与DA相互作用的化学计量比、作用位点和Kd。结果显示：Aβ1-16与DA相互作用的化学计量比为1：1；DA的酚羟基与Ap的Tyr10结合；Kd为0.30μmpl·L-1。通过定量检测·OH的产生量发现,适当浓度的DA可以释放Aβ-Cu2+中的Cu2+,增大·OH的产生量,增强其细胞毒性。圆二色光谱数据初步说明,Ap与DA的相互作用有可能促进Aβ寡聚体的形成,加剧脑部的神经毒性。(4)SPR和电化学研究显示NE与Ap的相互作用机制与DA/Aβ的相似：化学计量比为1：1；NE的酚羟基与Ap的Tyr10结合；Kd为0.25μmol·L-1。通过定量检测·OH的产生量发现,适当浓度的NE可以释放A(3-Cu2+中的Cu2+,促进·OH的产生。圆二色光谱数据初步说明,Ap与NE的相互作用可能会促进Ap寡聚体的形成,加剧脑部局部区域的神经毒性。更多还原

【Abstract】 A(3is the major constitute of senile plaque, a hallmark in the brain of AD victims. It is well-known that Aβ is crucial for the development of AD. The interactions of Aβ with neurotransmitters (Cu DA and NE) may affect the neuron. And the interactions of A(3with Cu2+DA and NE are very important for the neurobiological processes and pathogenesis of AD, therefore, the mechanisms of A(3interacting with Cu2+, DA and NE are investigated to get the kinetic constants, and the roles of A(3interacting with Cu2+, DA and NE in the pathogenesis of AD are discussed preliminarily. Base on the above considerations, the following studies were carried out.(1) Interactions of Aβ with Cu2+are known to be pH-dependent and believed to play a crucial role in the neurotoxicity of AD. However, there is no reports about the affinity of Aβ-Cu2+under acidic pH. In this study, SPR sensor with immobilized Aβ was used to investigate the formation of Aβ-Cu2+complexes under acidic pH conditions. Dissociation constants (Kd) were calculated and shown to be pH-dependent, ranging from8.7×10-3to3.5×10-8mol·L-1under pH4.0-7.0. The physiological significance of Kd was preliminarily investigated by monitoring the generation of·OH in aerobic solutions containing Aβ-Cu2+and Cu2+. The results imply that acidic conditions could aggravate the oxidative stress in the presence of Cu2+, and the weak affinities of Aβ-Cu2+under acidic pH could further enhance the oxidative damage. However, under mildly acidic pH conditions, the formation of Ap-Cu2+reduced free Cu2+, inhibited the generation of·OH. In biological milieu, Aβ could protect neuron from oxidative stress.(2) Numerous studies in the recent ten years show a large variation on the dissociation constant of Aβ-Cu2+under different solvent conditions. Among the common buffers, Tris is considered to be the most reliable. However, Tris is rich of nucleophilic groups, so it may have a more diversified role in the interaction of Aβ with Cu2+. Under acidic conditions, a Tris-Aβ-Cu2+ternary complex was identified using ESI-MS and TEM. The results of surface plasmon resonance reveal that the formation of a ternary complex increases the dissociation constant by almost one order of magnitude. Consequently, toxicity assessment indicates that the generation of-OH induced by the Aβ-Cu2+complex is enhanced in the presence of Tris. Increased pH condition disfavors the formation of Tris-Ap-Cu2+complex.(3) Due to the controversial effects of DA on the toxicity of Aβ and the development of AD, it is necessary to understand the interaction of Aβ with DA. In this study, the stoichiometric ratio (1:1), binding site (Tyr10) and dissociation constant (KA=0.30μmol·L-1) of DA interacting with Aβ1-16were revealed. The physiological significance of Kd was investigated by monitoring the generation of·OH in aerobic solutions containing Cu2+. The results demonstrat that DA inhibits the interaction of Aβ with Cu2+, enhances the generation of-OH. The CD results preliminarily imply that DA may improve the formation of Aβ oligomer, and then enhance the neurotoxicity.(4) The stoichiometric ratio (1:1), binding site (Tyr10) and dissociation constant (Kd=0.25μmol·-L-1) of NE interacting with Aβwere revealed by electrochemistry and SPR techniques. The physiological significance of Kd was investigated by monitoring the generation of·OH in aerobic solutions containing Cu2+. The results demonstrate that NE inhibits the interaction of Aβ with Cu2+, enhances the generation of-OH. The CD results preliminarily imply that NE may improve the formation of Aβ oligomer.更多还原