【摘要】：背景：阿爾茨海默病(Alzheimer's disease, AD)是一種臨床表現(xiàn)為進(jìn)行性記憶認(rèn)知功能障礙的神經(jīng)退行性疾病,是最常見的老年期癡呆,且患病率日趨上升,給家庭和社會(huì)帶來了沉重的負(fù)擔(dān)。由于其病因和發(fā)病機(jī)制復(fù)雜,目前尚無特效藥。腦源性神經(jīng)營(yíng)養(yǎng)因子(Brain-derived neurotrophic factor, BDNF)是一類對(duì)多種神經(jīng)元均有保護(hù)作用的神經(jīng)營(yíng)養(yǎng)因子,可通過激活TrkB受體及其下游Erkl/2和Akt信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)神經(jīng)元的存活、增殖分化和損傷修復(fù)。由于BDNF分子量大不能透過血腦屏障和藥物半衰期較短等因素,大大制約了BDNF的臨床應(yīng)用。因此,我們?cè)诒狙芯恐袘?yīng)用具有穿透生物膜作用的TAT肽和模擬BDNF的核心片段組成一段融合多肽。 目的：研究由TAT肽和模擬BDNF的核心片段組成的融合多肽是否具有神經(jīng)保護(hù)和促進(jìn)學(xué)習(xí)記憶能力,并探討其作用機(jī)制。 方法：本實(shí)驗(yàn)首先用EGFP標(biāo)記TAT,檢測(cè)其透過血腦屏障的能力。然后分別在東莨菪堿所致癡呆的大鼠模型和APP轉(zhuǎn)基因小鼠模型中外周給予融合多肽,用水迷宮檢測(cè)動(dòng)物的學(xué)習(xí)記憶能力改變,通過免疫印跡和免疫組化測(cè)定突觸相關(guān)蛋白的表達(dá),并根據(jù)模型動(dòng)物的特征分別檢測(cè)了膽堿能相關(guān)的酶的活性(AChE、ChAT)和Aβ的水平,為探討機(jī)制我們進(jìn)一步檢測(cè)了TrkB受體和下游信號(hào)轉(zhuǎn)導(dǎo)分子Erkl/2、Akt及其磷酸化水平,同時(shí)檢測(cè)轉(zhuǎn)錄因子和即早基因的表達(dá)。 結(jié)果：我們發(fā)現(xiàn)(1)TAT具有攜帶大分子物質(zhì)穿透血腦屏障的能力。(2)融合多肽能逆轉(zhuǎn)由東莨菪堿所導(dǎo)致的大鼠癡呆,促進(jìn)突觸素和M受體的表達(dá)并降低AChE的活性,其機(jī)制為融合多肽激活TrkB受體,促進(jìn)下游Erkl/2、Akt信號(hào)轉(zhuǎn)導(dǎo)通路以及轉(zhuǎn)錄因子CREB的活化。(3)融合多肽改善APP轉(zhuǎn)基因小鼠的學(xué)習(xí)記憶能力障礙,減少Aβ的水平和tau蛋白的過度磷酸化,增加突觸相關(guān)蛋白PSD93、PSD95的表達(dá),其作用機(jī)制為融合多肽激活TrkB受體,啟動(dòng)下游Erkl/2、Akt信號(hào)轉(zhuǎn)導(dǎo)通路降低BACE1、PS1以及GSK-3β活性。 結(jié)論：融合多肽能透過血腦屏障,通過激活TrkB受體及下游信號(hào)轉(zhuǎn)導(dǎo)通路調(diào)節(jié)突觸可塑性,有效改善AD模型鼠的腦病理改變和學(xué)習(xí)記憶障礙。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16

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