Presenilins条件性双基因敲除小鼠超微结构的增龄性变化及运动能力的研究
发布时间：2018-12-17 21:38
【摘要】：阿尔茨海默症(Alzheimer's disease, AD)是最常见的中枢神经系统退行性疾病。随着年龄的增加,AD的发病率几乎呈指数增长,严重影响了老年人的健康和生活品质。大多数AD呈散发型,少数患者具有家族性(Familial Alzheimer's disease, FAD),为常染色体显性遗传。研究发现,分别位于14号及1号染色体上的Presenilinl(PS1)及Presenilin2(PS2)基因的突变是导致早发性FAD最主要的原因。用前脑特异性敲除PS1的小鼠与全身性敲除PS2的小鼠繁育出的PS1/2条件性双基因敲除(dKO)小鼠表现出：神经元大量凋亡、Tau蛋白的高度磷酸化、剧烈的中枢神经系统和外周神经系统的炎症反应和认知功能障碍等一系列类似AD的症状。 有研究证据表明PS1参与了突触的形成并且具有调节突触功能的作用。大脑记忆功能的实现是通过增强神经元之间的联系而产生的。细胞核和线粒体参与细胞凋亡途径。PS1基因敲除会阻碍细胞内v-ATPaseV0al亚基从内质网运输到溶酶体,影响了v型ATP酶在溶酶体膜上的组装,进而阻碍了溶酶体的降解功能。 AD的病理学特征主要表现为神经元大量凋亡、老年斑的增加和神经纤维缠结。越来越多的证据表明,AD有一个较长的临床前阶段。在临床前阶段,病人的病理学特征增加,机体功能下降。但是这些特征不足以确诊为痴呆。实际上导致AD的病理生理过程在AD临床确诊前几年甚至几十年已经渐渐发生。一些非认知相关的行为,如运动功能障碍,可提示随后的AD病程的发展。因此,这类运动功能障碍的行为可作为AD临床前阶段的表现特征。研究结果显示了,AD病人在临床前阶段或者临床后期均表现出运动行为异常。 本文主要研究Presenilins条件性双基因敲除对于小鼠海马与皮层超微结构的增龄性影响以及对运动平衡能力的影响。 1. Presenilins条件性双基因敲除对于小鼠海马与皮层超微结构的影响 选用3月龄、6月龄及12月龄Presenilins1/Presenilins2双敲除小鼠(dKO)和对照小鼠(CON),运用透射电子显微镜技术,分别观察海马与皮层突触、细胞核、线粒体、溶酶体超微结构的改变。结果显示3月龄dK0小鼠海马与皮层溶酶体已向具有消化能力的次级溶酶体转化。6月龄dKO小鼠海马与皮层的突触后致密物厚度减小；皮层细胞核膜内陷,核不规则；海马与皮层线粒体出现肿胀、脊变形或消失；出现高电子密度的次级溶酶体,并伴有脂褐素小体出现。12月龄dKO小鼠海马与皮层突触后致密物厚度减小；海马与皮层核膜内陷,染色质固缩沿核膜分布；线粒体严重受损,脊大范围溶解；出现较多次级溶酶体和脂褐素小体。 Presenilins条件性双基因敲除对小鼠海马与皮层有增龄性的病理影响,这些病理改变将为相关的药物评价和对阿尔茨海默病的深入研究提供形态学依据。 2. Presenilins条件性双基因敲除对于小鼠运动平衡能力的影响 选用2月龄、6月龄及12月龄Presenilins1/Presenilins2双敲除小鼠(dKO)和对照小鼠(CON),以行为学实验为手段,分别从旷场实验、转棒实验、平衡木实验和衣架实验来全面的观察dKO小鼠的运动平衡能力。结果显示自2月龄起dKO小鼠出现轻微的运动功能障碍。这也从另一个方面证实dKO小鼠是较好的AD模型。同时对dKO小鼠全面的运动功能研究也为了更深入的研究其认知或非认知行为奠定了基础。 3. Presenilins条件性双基因敲除对于小鼠运动平衡能力的影响的分子机制研究 选用2月龄、6月龄及12月龄Presenilins1/Presenilins2双敲除小鼠(dKO)和对照小鼠(CON),使用荧光定量PCR技术检测了皮层、小脑和海马的calbindinD28k mRNA表达水平；使用Western blot技术检测检测了dKO小鼠的皮层、小脑和海马的calbindinD28k蛋白表达水平。结果显示dK0小鼠的皮层、小脑和海马的calbindinD28k mRNA和蛋白水平与CON小鼠相比均无显著性差异。同时dK0小鼠和CON小鼠的calbindinD28k mRNA和蛋白在小脑部位的表达量显著高于皮层和海马部位。 该结果表明dK0小鼠出现运动功能障碍并不是通过calbindinD28k起到调节作用的,且calbindinD28k在小鼠小脑部位有高表达水平。
[Abstract]:Alzheimer's disease (AD) is the most common degenerative disease of the central nervous system. With the increase of age, the incidence of AD is almost exponential, which seriously affects the health and quality of life of the elderly. Most of the AD is sporadic, and a few patients have familial Alzheimer's disease (FAD), which is autosomal dominant inheritance. It was found that the mutation of Presenilinl (PS1) and Presenilin2 (PS2) gene on chromosome 14 and chromosome 1 was the main cause of early FAD. The PS1/ 2-conditioned two-gene knockout (dKO) mice bred with the mouse of PS1-specific knockout PS1 and the mouse of the systemic knockout PS2 showed a significant amount of neuronal apoptosis, the high phosphorylation of the Tau protein, Severe central nervous system and peripheral nervous system inflammation and cognitive dysfunction, a series of symptoms like AD. There is evidence that PS1 is involved in the formation of synapses and has the function of regulating synapses Effect. The brain memory function is realized by enhancing the contact between the neurons. The nuclear and the mitochondria are involved in the apoptosis of the cells. pathway. PS1 knockout will block the transport of the intracellular v-CaseVal subunit from the endoplasmic reticulum to the lysosome, which affects the assembly of the v-type ATP enzyme on the lysosome membrane, thus preventing the degradation of lysosomes. The pathology of AD is mainly characterized by a large number of neurons, an increase of senile plaque and a god. There is a growing number of evidence that there is a longer AD in AD Preclinical stage. At the pre-clinical stage, the pathology features of the patient are increased, and the machine The body function is down. But these features are not sufficient The diagnosis of dementia. In fact, the pathophysiological process of AD has been in the past few years or even decades prior to the diagnosis of AD. It is gradually taking place. Some non-cognitive-related behaviors, such as motion dysfunction, can prompt the subsequent AD The development of the course of the disease is, therefore, the behavior of this type of motor dysfunction can be used as the pre-clinical stage of AD The results of the study showed that the AD patients were in the pre-clinical stage or in the post-clinical stage. In this paper, the effects of Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse and the effect of the Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse are studied in this paper. The effect of dynamic balance capacity. 1. Presenilins conditional double gene knockout in mice The effects of the ultrastructure of the hippocampus and the cortex were 3 months, 6 months and 12 months of Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON). the synapse and the nucleus of the horse and the cortex, The changes of the ultrastructure of the mitochondria and lysosomes in the hippocampus and cortex of dK0 mice at the age of 3 months have been transformed into secondary lysosomes with digestive ability. The thickness of the postsynaptic density of the hippocampus and the cortex of the dKO mice at 6 months of age is reduced, and the nuclear membrane of the cortex and the nucleus do not have the rules. in that hippocampus and cortex of the 12-month-old dKO mice, the thickness of the dense matter in the hippocampus and the cortex of the cortex of the 12-month-old dKO mice was reduced, and the nuclei of the hippocampus and the cortex of the cortex and the chromatin condensation were distributed along the nuclear membrane; The mitochondria are severely damaged, and the ridge is dissolved in a large extent; it is shown that There are many levels of lysosomes and lipofuscin. Presenilins conditional double gene knock-out has a pathological effect on the aging of the hippocampus and the cortex of the mouse, and these pathological changes will be relevant drug evaluation and control. The in-depth study of Alzheimer's disease provides a morphological basis. 2. Presenilins Two-month, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON) were selected for the effects of conditional double gene knockout on the exercise balance of mice The experiment of behavior experiment, the experiment of rotating rod, the experiment of the balance wood and the experiment of the balance wood were carried out. and the exercise balance ability of the dKO mice is comprehensively observed by the experiment of the clothes rack and the clothes hanger. Mild exercise dysfunction in dKO mice from 2 months of age was shown. This also demonstrated that dKO mice were better AD models from another aspect, while the overall exercise function of dKO mice was studied. It also laid the foundation for a more in-depth study of its cognitive or non-cognitive behavior. 3. Presenilins The molecular mechanism of the effect of the two-gene knockout on the balance of mice's motion was selected from 2-month-old, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double-knockout mice (dKO) and control mice (CON), and the skin was detected using the fluorescence quantitative PCR technique. The expression level of calbindin D28k in the layer, cerebellum and hippocampus was detected by Western blot. calbindin D28k protein expression level in the cortex, cerebellum, and hippocampus. The results showed that calbini in the cortex, cerebellum and hippocampus of dK0 mice nD28k mRNA and protein levels were not significantly different from that of CON mice, while calbindinD2 of dK0 mice and CON mice The expression of 8k mRNA and protein in the cerebellum was significantly higher than that of the cortex and the hippocampus. The results showed that the dK0 mice had a motor dysfunction and not adjusted by calbindin D28k.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16
本文编号：2384832
[Abstract]:Alzheimer's disease (AD) is the most common degenerative disease of the central nervous system. With the increase of age, the incidence of AD is almost exponential, which seriously affects the health and quality of life of the elderly. Most of the AD is sporadic, and a few patients have familial Alzheimer's disease (FAD), which is autosomal dominant inheritance. It was found that the mutation of Presenilinl (PS1) and Presenilin2 (PS2) gene on chromosome 14 and chromosome 1 was the main cause of early FAD. The PS1/ 2-conditioned two-gene knockout (dKO) mice bred with the mouse of PS1-specific knockout PS1 and the mouse of the systemic knockout PS2 showed a significant amount of neuronal apoptosis, the high phosphorylation of the Tau protein, Severe central nervous system and peripheral nervous system inflammation and cognitive dysfunction, a series of symptoms like AD. There is evidence that PS1 is involved in the formation of synapses and has the function of regulating synapses Effect. The brain memory function is realized by enhancing the contact between the neurons. The nuclear and the mitochondria are involved in the apoptosis of the cells. pathway. PS1 knockout will block the transport of the intracellular v-CaseVal subunit from the endoplasmic reticulum to the lysosome, which affects the assembly of the v-type ATP enzyme on the lysosome membrane, thus preventing the degradation of lysosomes. The pathology of AD is mainly characterized by a large number of neurons, an increase of senile plaque and a god. There is a growing number of evidence that there is a longer AD in AD Preclinical stage. At the pre-clinical stage, the pathology features of the patient are increased, and the machine The body function is down. But these features are not sufficient The diagnosis of dementia. In fact, the pathophysiological process of AD has been in the past few years or even decades prior to the diagnosis of AD. It is gradually taking place. Some non-cognitive-related behaviors, such as motion dysfunction, can prompt the subsequent AD The development of the course of the disease is, therefore, the behavior of this type of motor dysfunction can be used as the pre-clinical stage of AD The results of the study showed that the AD patients were in the pre-clinical stage or in the post-clinical stage. In this paper, the effects of Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse and the effect of the Presenilins conditional double-gene knockout on the ultrastructure of the hippocampus and the cortex of the mouse are studied in this paper. The effect of dynamic balance capacity. 1. Presenilins conditional double gene knockout in mice The effects of the ultrastructure of the hippocampus and the cortex were 3 months, 6 months and 12 months of Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON). the synapse and the nucleus of the horse and the cortex, The changes of the ultrastructure of the mitochondria and lysosomes in the hippocampus and cortex of dK0 mice at the age of 3 months have been transformed into secondary lysosomes with digestive ability. The thickness of the postsynaptic density of the hippocampus and the cortex of the dKO mice at 6 months of age is reduced, and the nuclear membrane of the cortex and the nucleus do not have the rules. in that hippocampus and cortex of the 12-month-old dKO mice, the thickness of the dense matter in the hippocampus and the cortex of the cortex of the 12-month-old dKO mice was reduced, and the nuclei of the hippocampus and the cortex of the cortex and the chromatin condensation were distributed along the nuclear membrane; The mitochondria are severely damaged, and the ridge is dissolved in a large extent; it is shown that There are many levels of lysosomes and lipofuscin. Presenilins conditional double gene knock-out has a pathological effect on the aging of the hippocampus and the cortex of the mouse, and these pathological changes will be relevant drug evaluation and control. The in-depth study of Alzheimer's disease provides a morphological basis. 2. Presenilins Two-month, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double knockout mice (dKO) and control mice (CON) were selected for the effects of conditional double gene knockout on the exercise balance of mice The experiment of behavior experiment, the experiment of rotating rod, the experiment of the balance wood and the experiment of the balance wood were carried out. and the exercise balance ability of the dKO mice is comprehensively observed by the experiment of the clothes rack and the clothes hanger. Mild exercise dysfunction in dKO mice from 2 months of age was shown. This also demonstrated that dKO mice were better AD models from another aspect, while the overall exercise function of dKO mice was studied. It also laid the foundation for a more in-depth study of its cognitive or non-cognitive behavior. 3. Presenilins The molecular mechanism of the effect of the two-gene knockout on the balance of mice's motion was selected from 2-month-old, 6-month-old and 12-month-old Presenilins1/ Presenilins2 double-knockout mice (dKO) and control mice (CON), and the skin was detected using the fluorescence quantitative PCR technique. The expression level of calbindin D28k in the layer, cerebellum and hippocampus was detected by Western blot. calbindin D28k protein expression level in the cortex, cerebellum, and hippocampus. The results showed that calbini in the cortex, cerebellum and hippocampus of dK0 mice nD28k mRNA and protein levels were not significantly different from that of CON mice, while calbindinD2 of dK0 mice and CON mice The expression of 8k mRNA and protein in the cerebellum was significantly higher than that of the cortex and the hippocampus. The results showed that the dK0 mice had a motor dysfunction and not adjusted by calbindin D28k.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16
【参考文献】
相关期刊论文 前3条
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2 ;Prolonged Alzheimer-like Tau Hyperphosphorylation Induced by Simultaneous Inhibition of Phosphoinositol-3 Kinase and Protein Kinase C in N2a cells[J];Acta Biochimica et Biophysica Sinica;2005年05期
3 吴馥梅，杜红燕，，章子贵;突触界面曲率及其生理意义[J];神经解剖学杂志;1994年01期
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