孕晚期母体暴露脂多糖对中年子鼠认知功能影响及机制的研究

发布时间：2018-03-24 11:12

本文选题：细菌脂多糖　切入点：行为学　出处：《安徽医科大学》2013年博士论文

【摘要】：背景阿尔茨海默病(Alzheimer's disease, AD)是临床常见的慢性进展性神经系统变性疾病,其临床表现以学习和记忆障碍最为突出,被认为是病理性衰老的一种。散发性AD (Sporadic Alzheimer disease, SAD)占AD患者中的绝大部分,且随着年龄增长发病率呈指数增加。虽然目前关于SAD的始动和持续进展机制未明,但是主流观点认为SAD的发病可能涉及遗传和环境双重作用机制,其中环境因素可能起着更为重要的作用。越来越多的流行病学调查显示许多成年慢性疾病起源于人类生命早期(尤其胚胎期)的不良因素暴露,其中感染为最常见的不良因素之一。细菌脂多糖(Lipopolysaccharides, LPS)是公认的促炎剂。不少动物实验研究证据表明胚胎期暴露LPS可能会引起成年认知功能减退及其相关疾病的发生。这些研究虽然较为全面地评估了妊娠期母体暴露LPS对子代出生后生长发育及成年期神经系统成熟情况,但是大多数研究只观察至成年期认知行为学改变,且极少探讨其涉及的病理生理机制。因此,我们拟用封闭群母鼠暴露LPS来观察子鼠出生后直至中年期的生长发育情况、行为学改变和衰老和/或AD相关性病理生理改变,为AD的胎源发生假说提供实验依据。目的探讨AD的胎源学说。方法 ①取18只受孕CD-1小鼠随机分为两组。LPS组小鼠在孕15-17天每天经腹腔注射LPS (50μg/kg)。对照组小鼠同期经腹腔注射等容积生理盐水。经正常的孕期分娩和哺乳期,从每窝随机各取2只子鼠(雌雄各半),每组各得18只。记录从4周龄到33周龄的动态体重。分别于35日龄、290日龄和400日龄时进行相关的行为学实验检测(包括种属行为、感觉运动平衡能力、焦虑行为和自发探索活动能力、空间和非空间学习记忆能力)。②完成行为学评估后处死小鼠,分离海马,制作蜡块和组织芯片,采用免疫组织化学技术检测两组背侧海马DG区和CA1区各亚层是否具有AD最突出的病理生理改变—Ap负荷增加。③分离颞枕顶叶皮质,匀浆后提取上清液采用ELISA法检测两组脑内SOD、MDA和GSH-PX水平的改变。④处死小鼠前,摘眼球取血,离心后提取上清液采用放射免疫法检测FT3和FT4水平。⑤采用免疫组织化学技术检测两组背侧海马各区域各亚层突触结合蛋白(Syt1和Syt4)和类泛素化蛋白(SUMO-3)的表达。结果 ①LPS组和同龄对照组小鼠出生后4周至33周体重动态变化虽无变化,但雄鼠体重大于雌鼠。在子鼠35日龄时,两组间的焦虑行为和自发探索活动(旷场活动)、空间学习记忆(六臂辐射状水迷宫)能力均无差异。在子鼠290日龄时,两组间的种属行为(储藏、挖掘和筑巢)、感觉平衡运动能力(平衡木和紧绳)、焦虑行为和自发探索活动(旷场活动)、空间学习记忆(六臂辐射状水迷宫)能力均无差异；在子鼠400日龄时,两组间的感觉平衡运动能力(平衡木和紧绳)、焦虑行为和自发探索活动(旷场活动、高架十字迷宫和黑白巷)能力保持相对良好,但是出现了LPS组小鼠储藏和挖掘能力显著增高,筑巢、空间(Morris水迷宫)和非空间(新物体再认)学习记忆能力显著降低。②LPS组小鼠背侧海马DG区的门区和分子层、CA1区分子层和起层的Ap水平均显著升高,与小鼠空间学习记忆减退有不同程度的相关性。③LPS组小鼠较对照组脑内的MDA含量显著增加、SOD及GSH-PX活力显著下降、血清FT4水平显著升高,并且与小鼠空间学习记忆减退均有不同程度的相关性。④LPS组小鼠背侧海马DG区各亚层、CA1分子层和放射层的Syt1含量,以及CA1区分子层、放射层和起层的Syt4含量均较对照组显著升高,但是两组间的DG区和CA1区各亚层的SUMO-3含量则无明显差异。其中LPS组小鼠Syt1含量与小鼠空间学习记忆减退有一定的相关性。小结 ①孕晚期母鼠重复暴露低剂量LPS的子鼠可经历正常的生长发育和神经系统成熟期,但到中年期其学习记忆(空间和非空间)能力较同龄对照组出现加速衰退；②此时期脑内出现Aβ负荷、氧化应激产物和突触结合蛋白含量增加、血清甲状腺激素水平升高。这些改变均与加速损害的年龄相关性空间学习记忆有不同程度关联。我们的实验结果表明胚胎期暴露LPS极有可能参与了中年子鼠的认知功能加速衰退过程,甚至有可能对AD的易感性增加。
[Abstract]:background
Alzheimer's disease (Alzheimer's disease AD) is a clinical common chronic progressive neurodegenerative disease, the clinical manifestations in learning and memory disorders are the most prominent, is considered to be a kind of pathological aging. Sporadic AD (Sporadic Alzheimer disease, SAD) accounted for the vast majority of patients with AD, and with the increase of age the incidence rate increases exponentially. Although the current and continued progress on SAD initiating mechanism is unknown, but the mainstream view is that the incidence of SAD may be related to genetic and environmental dual mechanisms in which environmental factors may play a more important role. More and more epidemiological survey shows that many adult chronic diseases originated in the early human life (especially the embryo stage) exposure to adverse factors, including infection is one of the most common adverse factors. Lipopolysaccharide (Lipopolysaccharides, LPS) is a proinflammatory agent recognized many. The animal experiment research evidence that embryonic exposure to LPS may cause dysfunction and related diseases of adult cognitive occurrence. Although these researches comprehensively evaluated the prenatal exposure to LPS on postnatal and adult nervous system maturation, but most of the research is only observed in adulthood, cognitive behavioral changes, and rarely discuss the pathophysiological mechanism involved. Therefore, we intend to use closed group after exposure to LPS was observed until the middle period development of offspring after birth, behavioral changes and senescence and / or AD related pathophysiologic changes, so as to provide experimental basis for the occurrence of AD fetal origin hypothesis.
objective
To discuss the fetal theory of AD.
Method
A total of 18 pregnant CD-1 mice were randomly divided into two groups: group.LPS mice at day 15-17 of gestation day by intraperitoneal injection of LPS (50 g/kg). The control group of mice by intraperitoneal injection with same volume of saline. After normal pregnancy childbirth and lactation, from each litter and randomly sampled 2 mice (male and female) in each group, each had 18. Records from 4 weeks to 33 weeks of age. The dynamic weight respectively at the age of 35, 290 and 400 days of age related behavioral test (including species behavior, sensorimotor ability, anxiety behavior and spontaneous exploration activities, and non empty space between the ability of learning and memory). The complete evaluation of the behavior after the mice were killed, isolated thehippocampus, wax block and tissue microarray, immunohistochemistry was performed in hippocampal DG region and CA1 region of the dorsal group two in each sub layer is AD the most prominent pathophysiological changes Ap the increase of the load. The separation of temporal pillow Cortex homogenate supernatant was extracted using ELISA method to detect two groups of brain SOD, MDA and GSH-PX level changes. The mice were sacrificed, eyeball blood, extracted from the supernatant by radioimmunoassay to detect FT3 and FT4 levels after centrifugation. The immunohistochemistry was performed in two groups of back side of each sub layer of synaptotagmin (Syt1 and Syt4) and ubiquitin protein (SUMO-3) expression.
4 to 33 weeks of weight change of mice no dynamic control group LPS after birth and age, but males weight more than female rats. The rats at the age of 35 days, the anxiety behavior between the two groups and locomotor activity (open field), spatial learning and memory (six arm water maze) to force there were no differences in offspring. At the age of 290 days between the two groups of species (storage, mining and nesting behavior), sensation balance exercise capacity (the balance beam and the tight rope), anxiety behavior and locomotor activity (open field), spatial learning and memory (six arm water maze) to force no differences in offspring; at the age of 400 days, feel balanced movement ability between the two groups (the balance beam and the tight rope), anxiety behavior and locomotor activity (open field, elevated plus maze and black white alley) to maintain a relatively good ability, but there is a nest of mice in group LPS storage and mining capabilities increased significantly. (Morris, space Water maze) and non spatial (novel object recognition) learning and memory ability of mice in group LPS decreased significantly. The DG region of the dorsal hippocampus of the hilus and molecular layer, CA1 layer and layer of the molecular level of Ap increased significantly, and the spatial learning and memory deficits in mice have different degrees of relevance. The mice in group LPS compared with the control group the content of MDA in brain increased significantly, SOD and GSH-PX activity decreased significantly, the serum levels of FT4 increased significantly, and the correlation between learning and memory deficits in varying degrees. The mice in group LPS in the DG region of the dorsal hippocampus of each sub layer, the content of Syt1 CA1 molecular layer and radiation layer, and CA1 molecules the radiation layer and layer, layer of Syt4 were significantly higher than the control group, but there were no significant differences in each sub layer of the SUMO-3 content of DG and CA1 region between the two groups. The mice in group LPS and Syt1 content of mice spatial learning and memory deficits have certain relevance. Summary
The late pregnancy rats repeatedly exposed to low dose LPS rats can experience growth and nervous system of normal maturity, but to the middle period of the learning and memory (spatial and non spatial ability) than the control group in this period appears to accelerate the decline; brain A beta load, oxidative stress and synaptotagmin content increased, elevated serum thyroid hormone levels. These changes were correlated with the age of space accelerate damage to some extent associated with learning and memory. Our results suggest that embryonic exposure to LPS is likely involved in the process of cognitive function in middle-aged rats can accelerate the recession, there may even increase the susceptibility to AD.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

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