透脑屏障的模拟脑源性神经营养因子融合多肽的神经保护作用及机制研究

发布时间：2019-05-14 19:13

【摘要】：背景：阿尔茨海默病(Alzheimer's disease, AD)是一种临床表现为进行性记忆认知功能障碍的神经退行性疾病,是最常见的老年期痴呆,且患病率日趋上升,给家庭和社会带来了沉重的负担。由于其病因和发病机制复杂,目前尚无特效药。脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)是一类对多种神经元均有保护作用的神经营养因子,可通过激活TrkB受体及其下游Erkl/2和Akt信号转导通路促进神经元的存活、增殖分化和损伤修复。由于BDNF分子量大不能透过血脑屏障和药物半衰期较短等因素,大大制约了BDNF的临床应用。因此,我们在本研究中应用具有穿透生物膜作用的TAT肽和模拟BDNF的核心片段组成一段融合多肽。目的：研究由TAT肽和模拟BDNF的核心片段组成的融合多肽是否具有神经保护和促进学习记忆能力,并探讨其作用机制。方法：本实验首先用EGFP标记TAT,检测其透过血脑屏障的能力。然后分别在东莨菪碱所致痴呆的大鼠模型和APP转基因小鼠模型中外周给予融合多肽,用水迷宫检测动物的学习记忆能力改变,通过免疫印迹和免疫组化测定突触相关蛋白的表达,并根据模型动物的特征分别检测了胆碱能相关的酶的活性(AChE、ChAT)和Aβ的水平,为探讨机制我们进一步检测了TrkB受体和下游信号转导分子Erkl/2、Akt及其磷酸化水平,同时检测转录因子和即早基因的表达。结果：我们发现(1)TAT具有携带大分子物质穿透血脑屏障的能力。(2)融合多肽能逆转由东莨菪碱所导致的大鼠痴呆,促进突触素和M受体的表达并降低AChE的活性,其机制为融合多肽激活TrkB受体,促进下游Erkl/2、Akt信号转导通路以及转录因子CREB的活化。(3)融合多肽改善APP转基因小鼠的学习记忆能力障碍,减少Aβ的水平和tau蛋白的过度磷酸化,增加突触相关蛋白PSD93、PSD95的表达,其作用机制为融合多肽激活TrkB受体,启动下游Erkl/2、Akt信号转导通路降低BACE1、PS1以及GSK-3β活性。结论：融合多肽能透过血脑屏障,通过激活TrkB受体及下游信号转导通路调节突触可塑性,有效改善AD模型鼠的脑病理改变和学习记忆障碍。
[Abstract]:Background: Alzheimer's disease (Alzheimer's disease, AD) is a neurodegenerative disease with progressive memory and cognitive dysfunction. It is the most common dementia in the elderly, and the prevalence rate is increasing day by day. It brings a heavy burden to the family and society. Because of its complex etiology and pathogenesis, there are no specific drugs at present. Brain-derived neurotrophic factor (Brain-derived neurotrophic factor, BDNF) is a kind of neurotrophic factor which has protective effect on many kinds of neurons. It can promote the survival of neurons by activating TrkB receptor and its downstream Erkl/2 and Akt signal transduction pathways. Proliferation, differentiation and repair of injury. Because the molecular weight of BDNF can not penetrate the blood-brain barrier and the half-life of drugs is short, the clinical application of BDNF is greatly restricted. Therefore, we used TAT peptides with penetrating biofilm and core fragments of simulated BDNF to form a fusion polypeptide in this study. Aim: to study whether the fusion polypeptide composed of TAT peptide and mimic BDNF core fragment has neuroprotection and promote learning and memory ability, and to explore its mechanism. Methods: in this experiment, EGFP labeled TAT, was used to detect its ability to penetrate the blood-brain barrier (BBB). Then fusion peptides were given to the rat model of dementia induced by scopolamine and the model of APP transgenic mice, and the changes of learning and memory ability of the animals were detected by water maze. The expression of synaptic related proteins was detected by immunoblotting and immunohistochemistry, and the activities of cholinergic related enzymes (AChE,ChAT) and A 尾 were detected according to the characteristics of the model animals. In order to explore the mechanism, we further detected the levels of TrkB receptor and downstream signal transduction molecule Erkl/2,Akt and its phosphorylation, as well as the expression of transcription factors and early genes. Results: we found that (1) TAT had the ability to carry macromolecular substances through the blood-brain barrier. (2) Fusion peptides could reverse dementia induced by scopolamine, promote the expression of synaptophysin and M receptor and decrease the activity of AChE. The mechanism is that fusion polypeptide activates TrkB receptor, promotes downstream Erkl/2,Akt signal transduction pathway and activation of transcription factor CREB. (3) Fusion polypeptide improves learning and memory impairment in APP transgenic mice. It decreased the level of A 尾 and hyperphosphorylation of tau protein and increased the expression of synaptic related protein PSD93,PSD95. The mechanism was that fusion polypeptide activated TrkB receptor and activated downstream Erkl/2,Akt signal transduction pathway to decrease the activity of BACE1,PS1 and GSK-3 尾. Conclusion: fusion polypeptide can regulate synaptic plasticity through blood-brain barrier, activate TrkB receptor and downstream signal transduction pathway, and effectively improve brain pathological changes and learning and memory impairment in AD model mice.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

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