阿尔茨海默病模型病变过程中的免疫机制及运动干预

发布时间：2018-05-05 22:58

本文选题：阿尔茨海默症 + 小胶质细胞　；参考：《河北师范大学》2012年博士论文

【摘要】：阿尔茨海默症(Alzheimer's disease, AD)是一种不可逆的神经退行性疾病,其特征表现为记忆和认知功能的逐渐丧失。阿尔茨海默症通常可以分为两种类型：一是家族性AD,二是散发型AD,大多数患者为后者。后者通常在60岁之后才会出现明显的AD病征。因此,散发型AD是与衰老相关的一种神经退行性疾病。目前已经明确AD的病理特征为神经元内由于tau蛋白过度磷酸化形成的神经缠节、细胞间β-淀粉样蛋白(Aβ)沉积形成的老年斑。由小胶质细胞(microglia, MG)和星形胶质细胞(astrocytes, AS)过度激活引发的系列炎症反应被认为是AD的第三个病理特征和发病原因之一。神经炎症反应机制为：不溶性Aβ的聚集和纤维化激活了MG。激活的MG通过产生活性氧、补体成分、促炎症细胞因子、趋化因子等引发一系列的炎症反应,形成神经毒性,造成神经元死亡；产生的促炎症细胞因子又可激活AS,从而共同参与脑局部炎症反应,最终形成AD。但随着研究发现,在正常的老年人脑内也存在弥散的Ap聚集物。Aβ的沉积是脑老化的产物。还有研究发现MG的形态与年龄有关。随着老龄化,MG的形态变为营养不良状态。在tau-蛋白磷酸化位点和Aβ斑块周围同样也发现MG的营养不良状态。因此认为渐行性的MG老化和MG保护功能的丢失是AD发病的原因之一。AS在AD发病中的作用也有不同的观点。在慢性神经退行性疾病中,众多的研究表明AS可以做为具有固有免疫能力的细胞参与免疫识别、免疫调节和炎症过程。但也有研究发现AS也存在着营养不良状态。在转基因鼠中随着鼠龄的增加,远离Aβ斑块的AS表现出脱分枝,GFAP表达降低,呈现出营养不良状态。因此提出AD发病的AS老化假说,即围绕Aβ斑块的AS表现为胶质化,而远离Aβ斑块的AS为营养不良状态。因此,针对MG和AS在AD发病中的作用有两种截然不同的假说。一是MG和AS过度激活和胶质化引发的炎症反应是AD发病的原因之一,表现为脑部促炎症因子的增加和MG及AS的过度激活；二是MG和AS老化及保护功能丧失是AD发病的原因之一,表现为营养不良的MG和AS细胞形态。本研究针对上述两种假说,利用不形成老年斑的AD模型——加速衰老模型(accelerated senescence-prone mouse8, SAMP8)小鼠作为研究材料,利用水迷宫、免疫组织化学染色、PT-PCR、Western blo、luminex等技术和方法研究了SAMP8小鼠在AD发病进程中认知能力的改变,海马和皮层中MG和AS的形态、数量以及功能的变化,并研究了不同性别之间的差异。在此基础上研究了不同年龄阶段的锻炼对学习和记忆能力的干预作用及免疫机制。本研究结果表明,2、5月龄的雄、雌SAMP8小鼠认知能力均没有显著变化,但8月龄的小鼠认知能力显著下降,雌鼠认知损伤更为严重。2、5月龄小鼠海马和皮层中,MG和AS的形态为胞体肥大、分支较多,处于激活状态；8月龄小鼠的MG和AS的形态发生了显著的改变。MG周围组织开始出现空泡,胞体开始萎缩,分支出现断裂；有些AS胞体模糊,没有完整的胞体形态,分支减少；而且AS完整细胞的数量显著降低,在雌性小鼠海马和皮层中,AS数量降低更为明显。MG表面标记CDllb和AS表面标记GFAP表达也显著降低。本研究从营养功能、吞噬作用及促炎症因子和趋化因子分泌三个方面研究了MG和AS功能。第一,随着月龄增加,营养功能降低。8月龄的小鼠海马和皮层中的BDNF和GDNF表达显著降低,GDNF的蛋白水平显著降低。其中GDNF的表达和蛋白水平从5月龄就开始显著降低,而且雌性小鼠海马中GDNF的降低幅度比雄性小鼠更大。第二,随着月龄增加,吞噬功能降低。TLR2、TLR4和MyD88基因的表达在2、5月龄时没有显著性差异,但在8月龄时显著性降低。性别之间没有显著性差异。第三,随着月龄增加,促炎症因子和趋化因子水平增加。海马和皮层中的L-1β、TNF-α在2、5月龄时没有显著性差异,在8月龄时显著增加,而IFN-γ则没有显著性差异。趋化因子MCP-1的水平在5月龄时开始显著增加。MG是中枢神经系统(CNS)的免疫细胞,AS是支持细胞,其激活首先表现的是免疫修复和营养支持功能。正常的修复过程可能包括两个阶段。第一阶段是炎症反应阶段。MG和AS表现为激活状态,通过吞作用,产生促炎症因子,清理造成微损伤的异物或者细胞碎片,表现为炎症反应；随后进入第二阶段,修复阶段。此阶段,营养因子水平增加,炎症因子水平降低,损伤的神经元得到修复。SAMP8小鼠3月龄出现氧化应激,5月龄出现tau蛋白过度磷酸化,这些都会对脑部造成微损伤,从而激活MG和AS。我们的研究结果也表明2、5月龄的MG和AS为激活状态。但此时的MG和AS功能处于正常水平,所以能够完成自我修复的过程,所以不会造成促炎症因子的积累,同时在认知水平上也不会有显著的减低,我们的研究结果说明了这一点。随着小鼠老龄化,MG和AS开始老化,其吞噬功能降低,自我修复功能停留在第一阶段,炎症反应阶段,造成促炎症因子的积累；其次营养功能降低,分泌的营养因子水平降低,不能很好完成后续的修复,从而造成损伤的持续和扩大,最终造成神经元的死伤。同时趋化因子分泌的增加,进一步招募外周的吞噬细胞进入CNS,进一步加重了炎症反应。因此,根据上述研究结果, 我们认为在小鼠老化过程中,MG和AS表现出相应的老化,其保护功能相应降低。到目前为止还没有有效的药物治疗AD,而运动锻炼提供了唯一不使用药物的干预措施。我们的研究结果表明,小鼠不同年龄阶段的锻炼产生的效果不同。病理特征产生之前就开始的全程有规律的锻炼(2月龄到8月龄)和早期有规律的锻炼(2月龄到4月龄)对认知功能的提高有显著的作用,而病理出现之后的锻炼(6月龄到8月龄)只有延缓和改善作用。研究结果还表明锻炼对雌性小鼠的影响更显著。通过我们的研究发现锻炼预防AD的免疫机制为：锻炼延缓了MG和AS的衰老,阻止了AS数量的减少,从而保证了MG和AS的功能。具体表现为：增加了BDNF和GDNF的表达和GDNF的蛋白水平；增加了TLR2及Myd88的表达,提高了吞噬功能；提高了TNF-γ的水平,降低了促炎症细胞因子的水平。因此,我们认为体育锻炼延缓了神经胶质细胞的老化,保证了神经胶质细胞的功能。
[Abstract]:Blzheimer (Alzheimer's disease, AD) is an irreversible neurodegenerative disease characterized by a gradual loss of memory and cognitive function. Alzheimer's disease is usually divided into two types: one is familial AD, two is a scattered AD, and most patients are the latter. The latter usually occurs after 60 years of age. AD symptoms. Therefore, the diffuse AD is a neurodegenerative disease associated with senescence. It is now clear that the pathological features of AD are the neural tangle formed by the excessive phosphorylation of tau protein in neurons, and the senile plaques formed by the deposition of beta amyloid protein (A beta) between cells. Microglia (MG) and astrocytes (astroc). Ytes, AS) a series of inflammatory reactions induced by excessive activation is considered to be one of the third pathological features and causes of AD. The mechanism of neuroinflammatory reaction is that the aggregation and fibrosis of insoluble A beta activates MG. activated MG by producing a series of inflammatory reactions by producing living oxygen, complement components, and promoting inflammatory cell causes, chemokines, and so on. The formation of neurotoxicity causes neuronal death, and the resulting pro-inflammatory cytokines can activate AS to participate in the local inflammatory response of the brain and eventually form AD., but as the study found that the deposition of the dispersed Ap aggregation.A beta in the normal human brain is a product of brain aging. And the study found that the form of MG and the age of the MG have been found. With aging, the form of MG becomes malnutrition. The malnutrition of MG is also found around the tau- protein phosphorylation site and the A beta plaque. Therefore, the gradual MG aging and the loss of MG protection function are one of the reasons for the pathogenesis of AD. The role of.AS in the pathogenesis of AD is also different. In chronic neurodegenerative disease In the disease, many studies have shown that AS can be used as a cell with inherent immune ability to participate in immune recognition, immunomodulation and inflammatory processes. But there are also studies found that AS also has malnutrition. In transgenic mice, with the increase of the age of the rat, the AS, which is far away from the A beta plaque, shows debranching, the expression of GFAP is reduced, and a malnutrition form is present. Therefore, the AS aging hypothesis of AD is proposed, that is, the AS performance around A beta plaques is glial, while AS far away from A beta plaque is malnutrition. Therefore, there are two distinct hypotheses on the role of MG and AS in AD pathogenesis. The increase of pro-inflammatory factors and excessive activation of MG and AS; two is the aging of MG and AS and the loss of protective function is one of the reasons for the pathogenesis of AD, which shows the MG and AS cell morphology of malnutrition. This study aims at the above two hypotheses, using the AD model that does not form the senile plaque, the accelerated aging model (accelerated senescence-prone mouse8, SAMP8) as a research material, the mice used water maze, immunohistochemical staining, PT-PCR, Western blo, Luminex and other techniques and methods to study the changes in the cognitive ability of SAMP8 mice during the pathogenesis of AD, the morphology, quantity and function of MG and AS in the hippocampus and cortex, and study the differences between different sexes. The results of this study showed that the cognitive ability of 2,5 months old male and female SAMP8 mice was not significantly changed, but the cognitive ability of 8 month old mice decreased significantly, and the cognitive impairment of female mice was more serious in the hippocampus and the cortex of.2,5 month old mice, MG and AS. In 8 month old mice, the morphology of MG and AS had a significant change in the morphology of MG and AS, and the cell body began to appear vacuoles, the body began to atrophy and the branch broke; some AS cell bodies were blurred, there was no complete cell morphology, and the branch decreased significantly, and the number of AS intact cells decreased significantly in female, in female In the hippocampus and cortex of mice, the number of AS decreased more obviously than the.MG surface marker CDllb and AS surface labeling GFAP expression. This study studied the function of MG and AS from three aspects: nutritional function, phagocytosis, proinflammatory factors and chemokine secretion. First, with the increase of month age, the nutrition function reduced the hippocampus and skin of mice aged.8 months. The expression of BDNF and GDNF in the layer decreased significantly, and the protein level of GDNF decreased significantly. The expression of GDNF and protein level began to decrease significantly from 5 month old, and the decrease of GDNF in the hippocampus of female mice was greater than that of male mice. Second, the phagocytic function decreased as the month of age increased, and the expression of TLR4 and MyD88 genes was at the age of 2,5. There was no significant difference, but there was no significant difference at 8 month old. There was no significant difference between sex. Third, the levels of pro-inflammatory and chemokines increased with the increase of month's age. There was no significant difference between L-1 beta and TNF- alpha in the hippocampus and cortex at the age of 2,5, and there was a significant increase at 8 month old, while IFN- gamma had no significant difference. Chemokines were not. The level of MCP-1 increases significantly at 5 month old when.MG is an immune cell of the central nervous system (CNS), and AS is a supporting cell. Its activation is the first expression of immune repair and nutritional support. The normal repair process may include two stages. The first stage is the activation state of the inflammatory response order.MG and AS, through the swallowing, and production. To promote inflammatory factors, clean up the foreign bodies or cell fragments that cause microdamage, and appear to be inflammatory reactions; then enter the second stage, repair stage. In this stage, the level of nutrition factor increases, the level of the inflammatory factors is reduced, the injured neurons are repaired.SAMP8 mice 3 month old out of oxidative stress, and 5 month old of the tau protein overphosphorylation, Some of the brain causes slight damage to the brain, thus activating MG and AS.. Our results also indicate that MG and AS are activated at 2,5 months, but the MG and AS functions at this time are at normal level, so it is possible to complete the process of self repair, so that there is no accumulation of pro-inflammatory factors and no significant reduction in cognitive levels, The results of our study show that as the aging of mice, the aging of MG and AS, its phagocytosis, the function of self repair stay in the first stage, the inflammatory reaction stage, the accumulation of pro-inflammatory factors; secondly, the decrease of nutritional function and the level of secreted nutrient factors, which can not complete the subsequent repair, thus making a good result. The persistence and enlargement of damage eventually causes the death and injury of neurons, and the increase of chemokine secretion and further recruitment of the peripheral phagocytes into CNS, which further aggravates the inflammatory response. Therefore, according to the results of these studies, we believe that in the aging process of mice, MG and AS show corresponding aging and their protective functions are correspondingly reduced. So far, there is no effective drug treatment for AD, and exercise provides the only intervention that does not use drugs. Our results show that the effects of exercise at different stages of the mice are different. Regular exercise (2 month old to 8 month old) and early regular exercise started before the pathological features were produced. (2 month old to 4 month old) had a significant effect on the improvement of cognitive function, and the effect of exercise (6 month old to 8 month old) after pathological appearance (6 month old to 8 month old) was only delayed and improved. The results also showed that exercise had more significant effects on female mice. Through our study, we found that exercise prevention of AD's immune mechanism was that exercise delayed the aging of MG and AS. The decrease in the number of AS has been stopped, which ensures the function of MG and AS, which is characterized by increasing the expression of BDNF and GDNF and the protein level of GDNF, increasing the expression of TLR2 and Myd88, improving the phagocytosis, improving the level of TNF- gamma, and reducing the level of pro-inflammatory cytokines. Therefore, we think that physical exercise has delayed the neuroglue. The aging of the mass cells ensures the function of the glial cells.

【学位授予单位】：河北师范大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

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