经鼻吸入胰岛素对阿尔茨海默病的作用以及机制研究

发布时间：2018-05-04 04:33

本文选题：阿尔茨海默病 + APP/PS1小鼠　；参考：《浙江大学》2016年博士论文

【摘要】：阿尔茨海默病(Alzheimer's disease, AD)是老年期痴呆最常见的病因,主要特点是进行性的认知和行为功能的损害。尽管全世界很多科学家都在致力于研究AD的病因,发病机制以及治疗方法,但目前仍然没有有效的可以治疗或者使疾病停止恶化的药物。有研究发现AD脑内存在胰岛素信号通路的缺陷,AD病人脑中胰岛素受体(insulin receptor, IR)的表达和结合能力都有异常。而且用淀粉样蛋白(amyloid beta, Aβ)寡聚体可以通过降低神经元表面的IR或与胰岛素竞争受体的方式阻断胰岛素信号通路向细胞内的传递。目前认为AD的胰岛素信号通路缺陷可能与散发性AD的发生相关。小范围的临床试验表明经鼻吸入胰岛素不仅可以改善健康受试者的注意力和记忆水平,对轻度认知障碍(mild cognitive impairment,MCI)和AD病人的记忆也有改善作用。但是胰岛素究竟是如何在AD病人的脑中发挥作用从而改善认知功能的分子机制目前仍然不清楚。我们前期的研究发现,对9月龄的三转基因AD小鼠(3xTg-AD mice)经鼻吸入胰岛素治疗1周后,可以修复胰岛素信号通路的缺陷,增加脑内突触蛋白的水平,降低脑内Aβ40的含量,减少脑内小胶质细胞的激活。为了更进一步地研究经鼻吸入胰岛素对早期AD病理改变的影响,我们采用4.5月龄的APP/PS1双转基因AD小鼠模型(APPswe/PS1dE9, APP/PS1),经鼻吸入胰岛素1U/天给药6周后,对AD模型小鼠的基本生理状况,行为学,分子生物学等进行了一系列的探索,主要的研究方法和结果如下：1.我们将所有的小鼠分成三组：APP/PS1胰岛素组(APP/PS1-ins), APP/PS1生理盐水组(APP/PS1-veh)和野生型生理盐水组(WT-veh)。给药前和给药后每周对这些小鼠监测体重,发现给胰岛素组与给生理盐水对照组以及非转基因小鼠相比体重的变化无明显差异。2.连续给药6周后,为评估三组小鼠的焦虑状态以及认知功能的情况,我们做了旷场实验(Open Field, OF)和水迷宫测试(Morris Water Maze, MWM). APP/PS1小鼠与WT小鼠比焦虑水平增高,经鼻吸入胰岛素后能降低该组小鼠的焦虑状态到WT小鼠的水平；在MWM实验的获得性训练(Acquisition Phase)阶段,三组小鼠的逃避潜伏期(Escape latency)和从入水点到平台的游泳距离(Path length to platform)无显著差异；在探查测验阶段,各组小鼠在原来平台所在象限所花的时间百分比也没有显著差别,这些结果表明6月龄的APP/PS1小鼠的窖认知功能在水迷宫测试中还未表现出明显的损害。但在反向迷宫实验(Reverse Morris Water Maze, rMWM)中,APP/PS1-veh小鼠的逃避潜伏期和游泳距离都比野生小鼠升高,胰岛素给药后有改善,提示APP/PS1小鼠已经表现出了记忆行为的可塑性异常,胰岛素对此有一定改善作用。3.免疫印迹方法分析三组小鼠的海马组织中胰岛素信号通路相关蛋白的表达水平,APP/PS1小鼠海马胰岛素信号通路相关蛋白的表达异常,经鼻吸入胰岛素可以改善这些蛋白表达的异常。与此结果相对应,c-Jun N-terminal kinase (JNK),这个在胰岛素抵抗和AD的病理学中发挥重要作用的激酶在APP/PS1小鼠海马中表达升高,经鼻吸入胰岛素降低JNK蛋白的磷酸化水平。4.免疫组化分析APP/PS1小鼠脑内的淀粉样蛋白斑块(Amyloid βplaque, Aβ plaque)的沉积,APP/PS1脑内存在明显的斑块沉积,统计分析提示经鼻吸入胰岛素可以减少APP/PS1小鼠皮层和海马的淀粉样斑块数量和面积。斑点印记(Dotblotting)实验发现经鼻吸入胰岛素减轻APP/PS1小鼠海马可溶性Ap寡聚体的含量。酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)结果显示胰岛素可以降低APP/PS1小鼠海马可溶性Aβ40 and Aβ42的水平。5.为了进一步研究胰岛素是否影响蛋白淀粉样前体蛋白(Amyloid precursor protein, APP)的剪切或影响APP蛋白的磷酸化从而使Ap的量发生变化,我们对APP蛋白的磷酸化水平以及APP加工过程中的中间产物做了免疫印迹分析。统计分析表明经鼻吸入胰岛素增加海马sAPPα的含量,减少sAPPβ的含量,并且使CTFβ/CTFα的比例下降,但不影响APP磷酸化蛋白的水平。6.APP的剪切和Ap的代谢过程中有许多酶的参与,包括p位点APP剪切酶1(Beta-secretase 1, BACE 1),主要的α位点剪切酶ADAM10,以及与Ap的降解和清除有关的IDE, APOE和LRP1等,APP/PS1小鼠海马BACE1和APOE水平与野生型小鼠相比升高,经鼻吸入胰岛素降低BACE1和APOE的表达水平,升高ADAM10的表达,但对IDE和LRP1没有明显影响。7. APP/PS1小鼠脑内星型胶质细胞和小胶质细胞的标志蛋白表达均增加,但没有发现胰岛素对此有明显的改善作用。与突触相关的三个蛋白突触前蛋白Synaptophysin,突触后密度蛋白95 PSD-95,神经突触素1Synapsin 1在三组小鼠脑中的表达水平无显著差异。8.6月龄APP/PS1小鼠海马Tau蛋白,磷酸化Tau蛋白的水平无明显异常；对与Tau磷酸化相关的几个激酶的免疫印迹分析也没有发现表达水平的异常。9.胰岛素增加APP/PS1小鼠海马内主要的神经元发生标志蛋白doublecortin的水平。终上所述,6月龄的APP/PS1转基因小鼠在水迷宫实验中虽然未表现出明显的认知功能障碍,但在反向迷宫测试中已经表现出了记忆可塑性的损害,经鼻吸入胰岛素对此有改善作用。APP/PS1小鼠焦虑水平升高,吸入胰岛素可缓解小鼠的焦虑水平。对小鼠海马的生化分析显示APP/PS1小鼠脑内胰岛素信号通路存在异常,胰岛素部分改善该信号通路的异常,可能与胰岛素能减轻小鼠脑内的JNK蛋白激活有关。转基因小鼠海马可溶性的Aβ40和A肛2水平,可溶性Ap寡聚体的含量,以及脑内淀粉样斑块的沉积均表现出不同程度的升高。吸入胰岛素后改善了脑内的Ap病理变化。进一步的研究发现胰岛素可能是通过调节APP蛋白的剪切加工,使APP蛋白的剪切更多地朝着不产生Ap的途径进行,并恢复脑内升高的APOE蛋白水平来减轻Ap病理变化。经鼻吸入胰岛素还对脑内神经元的发生有促进作用。
[Abstract]:Alzheimer's disease (AD) is the most common cause of Alzheimer's disease, characterized by progressive cognitive and behavioral impairment. Although many scientists around the world are devoted to the study of the etiology, pathogenesis and treatment of AD, there is still no effective treatment or cessation of the disease. Studies have found that the AD brain is deficient in the insulin signaling pathway, and the expression and binding ability of the insulin receptor (insulin receptor, IR) in the brain of AD patients is abnormal. And the amyloid (amyloid beta, A beta) oligomer can block the islets by reducing the IR on the surface of the neuron or by the insulin competition receptor. It is believed that the deficiency of AD's insulin signaling pathway may be associated with the occurrence of sporadic AD. Small range of clinical trials suggest that inhaled intranasal insulin can not only improve the level of attention and memory of healthy subjects, but also the memory of mild cognitive impairment (mild cognitive impairment, MCI) and AD patients. However, it is still unclear how insulin plays a role in the brain of AD patients and improves the molecular mechanism of cognitive function. Our previous study found that 9 month old of the three transgenic AD mice (3xTg-AD mice) could repair the defects of the insulin signaling pathway after 1 weeks of nasal inhalation of insulin. Increase the level of synapse protein in the brain, reduce the content of A beta 40 in the brain and reduce the activation of microglia in the brain. In order to further study the effect of nasal inhalation of insulin on the early pathological changes of AD, we used the 4.5 month old APP/PS1 double transgenic mouse model (APPswe/ PS1dE9, APP/PS1), and administered the nasal inhalation insulin for 6 weeks at 1U/ days. After that, a series of studies were carried out on the basic physiological status, behaviourology and molecular biology of AD model mice. The main methods and results were as follows: 1. we divided all the mice into three groups: APP/PS1 insulin group (APP/PS1-ins), APP/PS1 saline group (APP /PS1-veh) and wild type saline group (WT-veh). The weight of the mice was monitored every week, and there was no significant difference in weight between the insulin group and the normal saline control group and the non transgenic mice. After 6 weeks of.2. continuous administration, we made an open field experiment (Open Field, OF) and water maze test (Morri) to evaluate the anxiety state and cognitive function of the mice (Morri S Water Maze, MWM). The anxiety level of APP/PS1 mice and WT mice was higher than that of WT mice. After inhalation of insulin, the mice could reduce the anxiety state of the mice to the level of WT mice; the escape latency (Escape latency) and the swimming distance from the water entry point to the platform of the three groups in the MWM experiment acquired training (Acquisition Phase) stage. There was no significant difference in GTH to platform, and there was no significant difference in the percentage of time spent in the quadrant of the original platform at the exploratory test stage. These results showed that the cellar cognitive function of the 6 month old APP/PS1 mice did not show obvious damage in the water maze test. But in the reverse labyrinth test (Reverse Morris Water M) In aze, rMWM), the escape latency and swimming distance of APP/PS1-veh mice were higher than those in the wild mice, and the insulin was improved after the administration of insulin, suggesting that the APP/PS1 mice had shown the malfunction of the memory behavior, and the insulin was improved by the.3. immunoblotting method to analyze the insulin signaling pathway in the hippocampus of the three groups of mice. The expression level of related proteins, the abnormal expression of insulin signaling pathway related proteins in the hippocampus of APP/PS1 mice, can improve the abnormal expression of these proteins through the nasal inhalation of insulin. Corresponding to this result, c-Jun N-terminal kinase (JNK), a kinase that plays an important role in the insulin resistance and AD pathology, is in the APP/PS1 mice sea. The expression in the horse was elevated and the JNK protein phosphorylation level was reduced by the nasal inhalation of insulin.4. immunohistochemical analysis of the amyloid plaques (Amyloid beta plaque, A beta plaque) in the brain of APP/PS1 mice. The APP/PS1 brain was deposited in the distinct plaque. Statistical analysis suggested that the inhalation of insulin through the nose could reduce the cortex and hippocampus of APP/PS1 mice. The number and area of the amyloid plaque. The Dotblotting experiment found that the nasal inhalation of insulin alleviated the content of soluble Ap oligomers in the hippocampus of APP/PS1 mice. The results of enzyme linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) showed that insulin could lower the level of soluble A beta 40 and A beta 42 in the hippocampus of APP/PS1 mice. 5. in order to further study whether insulin affects the shear of protein amyloid precursor protein (Amyloid precursor protein, APP) or affects the phosphorylation of APP protein to change the amount of Ap, we have made an immunoblotting analysis of the phosphorylation level of the APP protein and the intermediate products in APP processing. Insulin content increased the content of sAPP alpha in the hippocampus, reduced the content of sAPP beta, and reduced the proportion of CTF beta /CTF alpha, but did not affect the shear of the APP phosphorylated protein level.6.APP and the participation of many enzymes during the metabolic process of Ap, including the APP shear enzyme 1 (Beta-secretase 1, BACE 1) at the P site, the main alpha site shear enzyme ADAM10, and the p Degradation and scavenging related IDE, APOE and LRP1, BACE1 and APOE levels in the hippocampus of APP/PS1 mice were higher than that of wild type mice. Nasal inhalation of insulin reduced the expression of BACE1 and APOE, increased the expression of ADAM10, but did not significantly affect the marker proteins of astrocytes and microglia in IDE and LRP1. The expression was increased, but it was not found that insulin had a significant improvement. Three synaptosomal precursor protein Synaptophysin, postsynaptic density protein 95 PSD-95, and synaptophysin 1Synapsin 1 in the three group of mice had no significant difference in the.8.6 month old APP/PS1 mice hippocampus Tau protein, the phosphorylated Tau protein. There was no obvious abnormality in the level; the immunoblotting analysis of several kinases associated with Tau phosphorylation also showed no expression level of abnormal.9. insulin that increased the level of the major neuronal marker protein Doublecortin in the hippocampus of APP/PS1 mice. Finally, 6 month old of the APP/PS1 transgenic mice were not shown in the water maze experiment. There is obvious cognitive impairment, but the impairment of memory plasticity has been shown in the reverse labyrinth test. The inhalation of insulin by nasal inhalation improves the anxiety level of.APP/PS1 mice, and inhaled insulin can relieve the anxiety level of mice. Biochemical analysis of the hippocampus of mice shows the insulin signaling pathway in the brain of APP/PS1 mice. The abnormal insulin partially improves the signal pathway, which may be related to the ability of insulin to reduce the activation of JNK protein in the brain of mice. The content of soluble A beta 40 and A anus, the content of soluble Ap oligomers, and the deposition of amyloid plaques in the brain are all elevated in different degrees. Ap pathological changes in the brain are good. Further studies have found that insulin may be by modulating the shearing process of APP protein to make APP protein cut more towards the non Ap pathway, and to restore the level of APOE protein in the brain to alleviate the pathological changes of Ap. Use.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.16

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