TREM2调节的小胶质细胞增殖与活化对AD病理的影响及作用机制

发布时间：2018-06-06 00:46

本文选题：TREM2 + PDTC　；参考：《广东药科大学》2017年硕士论文

【摘要】：阿尔茨海默病(Alzheimer’s Disease,AD),是一种进行性神经退行性疾病,其特征为细胞外淀粉样蛋白沉积和细胞内神经原纤维缠结。其临床症状主要表现为认知功能障碍和记忆减退,并伴有一系列精神和行为改变。AD发生的病因迄今不明,Aβ学说被公认为是AD发生的重要机制。该学说认为,由于代谢异常,导致脑内相关酶活性失调,产生有毒性作用的蛋白片段。该片段不能被及时清除,使其在脑内特定区域堆积,形成β淀粉样沉淀,进一步发展为老年斑,最终引起神经元凋亡,诱发阿尔茨海默病。β淀粉样蛋白(Aβ)是由39-43个氨基酸组成的构成老年斑的主要成分。Aβ40和Aβ42片段是淀粉样蛋白前体(APP)经蛋白水解酶水解后产生的最常见的β淀粉样蛋白片段,其中Aβ42片段含量较少,但是其更容易聚集形成淀粉样蛋白沉积,因此具有更强的毒性。小胶质细胞是脑内常驻免疫细胞,是中枢神经系统(CNS)免疫防线重要的构成细胞,在各种神经系统疾病以及炎症反应中具有极其重要的作用。生理状态下,活化的小胶质细胞在维持中枢神经系统正常功能中发挥重要的作用。II型髓系细胞触发型受体(TREM2)是小胶质细胞的表面受体,是小胶质细胞高度表达的与自身活化相关的蛋白,参与吞噬Aβ、修复受损神经元和清除细胞碎片的过程。近年来,基因组学研究发现,罕见的TREM2突变可显著增加阿尔茨海默病的发病机率。研究表明,TREM2缺失或单剂量不足都可导致AD模型鼠清除Aβ的能力明显下降。尽管基因突变可增加AD的发病机率,但是研究表明,年龄因素仍是AD发病的最主要因素。多种疾病研究发现,TREM2的表达受核转录因子NF-κB的调控。NF-κB是一类关键的核转录因子,几乎在所有类型细胞的胞质中都有以异源或同源二聚体非活性形式存在的NF-κB复合物,与机体代谢、基因转录翻译和调控蛋白质表达等生命活动密切相关。NF-κB作为细胞信号转导途径的枢纽,具有广泛的生物学活性,能够在基因水平上参与炎症、肿瘤、免疫、细胞增殖和细胞凋亡等多种生理、病理过程的调控。吡咯烷二硫代甲酸(PDTC)是一种可以透过细胞膜的特异性的NF-κB活化抑制剂,可以在多种细胞中抑制NF-κB的激活,产生相应的生物学活性。目的:本实验通过提高类AD模型小鼠脑内TREM2的表达,促进小胶质细胞的增殖活化,进一步检测活化的小胶质细胞对AD模型鼠病理过程的影响及其产生作用的机制。方法:1.脑立体定位注射Aβ1-42到昆明小鼠单侧海马CA1区制备类AD小鼠动物模型。对侧侧脑室注射不同浓度的PDTC。实验小鼠分为:空白组、Aβ组、PDTC处理组(6 mg/L、8 mg/L、600 mg/L、800 mg/L),注射后第7天取材。2.常规苏木素-伊红(HE)染色观察不同组中海马区损伤部位组织形态变化。3.免疫组织化学检测不同组海马内Aβ的沉积,免疫组织化学和Western Blot检测海马区TREM2、CD11B、BDNF和DCX的表达。4.免疫组织荧光技术检测海马区Aβ和小胶质细胞共表达、小胶质细胞和TREM2共表达。结果:1.HE染色结果显示:与空白组相比,注射Aβ各组小鼠海马区出现不同程度的损伤;Aβ组小鼠海马损伤区域最为明显,与Aβ组相比,给予PDTC处理后,PDTC各组海马区损伤明显减轻。2.Aβ免疫组织化学检测结果显示:空白组海马区没有Aβ沉积,注射Aβ后各组模型鼠海马区均有Aβ沉积;其中,Aβ组海马区Aβ沉积最多,PDTC处理后海马区Aβ沉积明显减少,结果具有显著性差异(P0.01)。3.TREM2免疫组织化学检测结果显示:与空白组相比,Aβ组海马区TREM2的表达降低,结果具有显著性差异(P0.01);PDTC处理后,TREM2表达增加,与Aβ相比,差异具有显著性(P0.01)。且在一定范围内,TREM2的表达与PDTC剂量呈正相关,;CD11B免疫组织化学结果显示,空白组海马区小胶质细胞均匀分布,无聚集现象;Aβ组和PDTC各组海马区小胶质细胞在Aβ周围集中分布;与Aβ组相比,PDTC各组小胶质细胞表达增加,结果具有显著性差异(P0.01)。在一定剂量范围内与PDTC给药剂量呈正相关。4.免疫组织荧光共定位检测结果显示:Aβ周围小胶质细胞大量聚集活化,Aβ沉积内有小胶质细胞的浸润;TREM2与小胶质细胞表达位置相关,具有共表达关系。5.BDNF、DCX免疫组织化学检测结果显示:与空白组相比,Aβ组BDNF和DCX的表达均有下降,PDTC各组BDNF和DCX的表达上升,差异具有显著性(P0.01)。在一定范围内,BDNF和DCX的表达与PDTC剂量具有依赖关系。结论:1.PDTC干预调节小鼠海马内TREM2的表达。2.TREM2表达增加,可激活小胶质细胞并维持特定小胶质细胞表型,从而清除Aβ和抑制炎症反应。3.活化的小胶质细胞可能分泌与神经再生相关的神经营养因子如BDNF,促进神经元再生,改善AD病理状态。
[Abstract]:Alzheimer 's Disease (AD) is an progressive neurodegenerative disease characterized by extracellular amyloid protein deposition and intracellular neurofibrillary tangles. Its clinical symptoms are cognitive dysfunction and memory impairment, accompanied by a series of mental and behavioral changes in.AD to date, A beta The theory is recognized as an important mechanism for the occurrence of AD. It is believed that the protein fragment of the brain related enzyme activity is caused by abnormal metabolism, which can not be removed in time to accumulate in certain regions of the brain, form beta amyloid precipitation, develop into senile plaques, eventually induce neuronal apoptosis and induce apoptosis. Beta amyloid (A beta) is the most common beta amyloid fragment produced by the hydrolysis of amyloid precursor (APP) by 39-43 amino acids, which is the main component of the aged plaque,.A beta 40 and A beta 42. The content of A beta 42 is less, but it is more easily aggregated to form amyloid eggs. White deposits are more toxic. Microglia, a resident immune cell in the brain, is an important constituent of the central nervous system (CNS) immune defense line. It plays an important role in various nervous system diseases and inflammatory reactions. Under physiological condition, activated microglia can maintain the normal function of the central nervous system. The important role of.II type myeloid cell contact cell receptor (TREM2) is the surface receptor of microglia, which is highly expressed by microglia and the proteins associated with self activation. It participates in the process of phagocytosis of A beta, repairing damaged neurons and scavenging cell debris. In recent years, the study of genomics has found that rare TREM2 mutations can be significantly increased. The incidence of Alzheimer's disease. Studies have shown that TREM2 deletion or single dose deficiency can lead to a significant decrease in the ability of AD model mice to clear A beta. Although genetic mutations can increase the incidence of AD, studies have shown that age is still the most important factor in the pathogenesis of AD. A variety of disease studies have found that the expression of TREM2 is subject to the nuclear transcription factor NF-. The regulation of kappa B.NF- kappa B is a key nuclear transcription factor. There are almost all types of NF- kappa B complex in the cytoplasm of all types of cells, which exist in the form of heterogeneous or homologous two polymer, and closely related to the body metabolism, gene transcription translation and the regulation of protein expression, which are closely related to.NF- kappa B as the hub of cell signal transduction pathway. PDTC is a specific NF- kappa B activation inhibitor that can inhibit the activation of NF- kappa B in a variety of cells and produces phase in a variety of cells. Objective: Objective: to improve the proliferation and activation of microglia by improving the expression of TREM2 in the brain of AD model mice, the effect of activated microglia on the pathological process of AD model rats and the mechanism of its effect were further detected. Method: 1. stereotactic stereotaxic injection of A beta 1-42 to the unilateral hippocampal CA1 area of Kunming mice AD mice were prepared by injecting different concentrations of PDTC. in the lateral ventricle. The mice were divided into blank group, A beta group, PDTC treatment group (6 mg/L, 8 mg/L, 600 mg/L, 800 mg/L), and seventh days after injection,.2. routine hematoxylin eosin (HE) staining was used to observe the changes of tissue morphology of the injured part of the CNOOC region. The deposition of A beta in the same group, immunohistochemistry and Western Blot for the detection of TREM2, CD11B, BDNF and DCX in the hippocampus, the expression of.4. immunofluorescence technique was used to detect the co expression of A beta and microglia in the hippocampus, and the co expression of microglia and TREM2. The damage area of the hippocampus in A beta group was most obvious. Compared with the A beta group, after PDTC treatment, the hippocampal damage in each group of PDTC was significantly reduced by.2.A beta immunochemistry test results showed that there was no A beta deposition in the hippocampus of the blank group and A beta deposition in the hippocampus of each model rat after the injection of A beta, and A beta deposition in the hippocampus of A beta group. At most, the A beta deposition in hippocampus decreased significantly after PDTC treatment. The results showed significant difference (P0.01). The results of.3.TREM2 immunohistochemical staining showed that the expression of TREM2 in the hippocampus of A beta group was significantly lower than that in the blank group (P0.01), and the expression of TREM2 increased after PDTC treatment, and the difference was significant (P0.01) compared with A beta. In a certain range, the expression of TREM2 was positively correlated with the dose of PDTC, and the results of CD11B immunohistochemical staining showed that the microglia in the hippocampus of the blank group was distributed uniformly and without aggregation, and the microglia in the hippocampus of A beta and PDTC groups was distributed around A beta, and the microglia expression in PDTC groups increased significantly compared with the A beta group, and the results were significant. Sex difference (P0.01). The results of positive correlation of.4. immunofluorescence with the dose of PDTC in a certain dose range showed that the microglia around A beta was activated in large quantities and there were microglia infiltration in A beta deposition; TREM2 was associated with the position of microglia expression, and there was a co expression relationship between.5.BDNF and DCX immuno histochemical examination. Compared with the blank group, the expression of BDNF and DCX in A beta group decreased, the expression of BDNF and DCX in PDTC groups increased, and the difference was significant (P0.01). In a certain range, the expression of BDNF and DCX was dependent on the PDTC dose. Conclusion: 1.PDTC intervention to regulate the expression of TREM2 in the hippocampus of mice increased and could activate small glue. The cells also maintain a specific microglia phenotype, thus removing A beta and inhibiting the activation of.3. activated microglia may secrete neurotrophic factors related to nerve regeneration, such as BDNF, promote neuron regeneration and improve the pathological state of AD.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

【相似文献】