發(fā)布時間：2018-03-24 11:12

  本文選題：細菌脂多糖　切入點：行為學　出處：《安徽醫(yī)科大學》2013年博士論文

【摘要】：背景 阿爾茨海默病(Alzheimer's disease, AD)是臨床常見的慢性進展性神經(jīng)系統(tǒng)變性疾病,其臨床表現(xiàn)以學習和記憶障礙最為突出,被認為是病理性衰老的一種。散發(fā)性AD (Sporadic Alzheimer disease, SAD)占AD患者中的絕大部分,且隨著年齡增長發(fā)病率呈指數(shù)增加。雖然目前關(guān)于SAD的始動和持續(xù)進展機制未明,但是主流觀點認為SAD的發(fā)病可能涉及遺傳和環(huán)境雙重作用機制,其中環(huán)境因素可能起著更為重要的作用。越來越多的流行病學調(diào)查顯示許多成年慢性疾病起源于人類生命早期(尤其胚胎期)的不良因素暴露,其中感染為最常見的不良因素之一。細菌脂多糖(Lipopolysaccharides, LPS)是公認的促炎劑。不少動物實驗研究證據(jù)表明胚胎期暴露LPS可能會引起成年認知功能減退及其相關(guān)疾病的發(fā)生。這些研究雖然較為全面地評估了妊娠期母體暴露LPS對子代出生后生長發(fā)育及成年期神經(jīng)系統(tǒng)成熟情況,但是大多數(shù)研究只觀察至成年期認知行為學改變,且極少探討其涉及的病理生理機制。因此,我們擬用封閉群母鼠暴露LPS來觀察子鼠出生后直至中年期的生長發(fā)育情況、行為學改變和衰老和/或AD相關(guān)性病理生理改變,為AD的胎源發(fā)生假說提供實驗依據(jù)。 目的 探討AD的胎源學說。 方法 ①取18只受孕CD-1小鼠隨機分為兩組。LPS組小鼠在孕15-17天每天經(jīng)腹腔注射LPS (50μg/kg)。對照組小鼠同期經(jīng)腹腔注射等容積生理鹽水。經(jīng)正常的孕期分娩和哺乳期,從每窩隨機各取2只子鼠(雌雄各半),每組各得18只。記錄從4周齡到33周齡的動態(tài)體重。分別于35日齡、290日齡和400日齡時進行相關(guān)的行為學實驗檢測(包括種屬行為、感覺運動平衡能力、焦慮行為和自發(fā)探索活動能力、空間和非空間學習記憶能力)。②完成行為學評估后處死小鼠,分離海馬,制作蠟塊和組織芯片,采用免疫組織化學技術(shù)檢測兩組背側(cè)海馬DG區(qū)和CA1區(qū)各亞層是否具有AD最突出的病理生理改變—Ap負荷增加。③分離顳枕頂葉皮質(zhì),勻漿后提取上清液采用ELISA法檢測兩組腦內(nèi)SOD、MDA和GSH-PX水平的改變。④處死小鼠前,摘眼球取血,離心后提取上清液采用放射免疫法檢測FT3和FT4水平。⑤采用免疫組織化學技術(shù)檢測兩組背側(cè)海馬各區(qū)域各亞層突觸結(jié)合蛋白(Syt1和Syt4)和類泛素化蛋白(SUMO-3)的表達。結(jié)果 ①LPS組和同齡對照組小鼠出生后4周至33周體重動態(tài)變化雖無變化,但雄鼠體重大于雌鼠。在子鼠35日齡時,兩組間的焦慮行為和自發(fā)探索活動(曠場活動)、空間學習記憶(六臂輻射狀水迷宮)能力均無差異。在子鼠290日齡時,兩組間的種屬行為(儲藏、挖掘和筑巢)、感覺平衡運動能力(平衡木和緊繩)、焦慮行為和自發(fā)探索活動(曠場活動)、空間學習記憶(六臂輻射狀水迷宮)能力均無差異；在子鼠400日齡時,兩組間的感覺平衡運動能力(平衡木和緊繩)、焦慮行為和自發(fā)探索活動(曠場活動、高架十字迷宮和黑白巷)能力保持相對良好,但是出現(xiàn)了LPS組小鼠儲藏和挖掘能力顯著增高,筑巢、空間(Morris水迷宮)和非空間(新物體再認)學習記憶能力顯著降低。②LPS組小鼠背側(cè)海馬DG區(qū)的門區(qū)和分子層、CA1區(qū)分子層和起層的Ap水平均顯著升高,與小鼠空間學習記憶減退有不同程度的相關(guān)性。③LPS組小鼠較對照組腦內(nèi)的MDA含量顯著增加、SOD及GSH-PX活力顯著下降、血清FT4水平顯著升高,并且與小鼠空間學習記憶減退均有不同程度的相關(guān)性。④LPS組小鼠背側(cè)海馬DG區(qū)各亞層、CA1分子層和放射層的Syt1含量,以及CA1區(qū)分子層、放射層和起層的Syt4含量均較對照組顯著升高,但是兩組間的DG區(qū)和CA1區(qū)各亞層的SUMO-3含量則無明顯差異。其中LPS組小鼠Syt1含量與小鼠空間學習記憶減退有一定的相關(guān)性。小結(jié) ①孕晚期母鼠重復(fù)暴露低劑量LPS的子鼠可經(jīng)歷正常的生長發(fā)育和神經(jīng)系統(tǒng)成熟期,但到中年期其學習記憶(空間和非空間)能力較同齡對照組出現(xiàn)加速衰退；②此時期腦內(nèi)出現(xiàn)Aβ負荷、氧化應(yīng)激產(chǎn)物和突觸結(jié)合蛋白含量增加、血清甲狀腺激素水平升高。這些改變均與加速損害的年齡相關(guān)性空間學習記憶有不同程度關(guān)聯(lián)。我們的實驗結(jié)果表明胚胎期暴露LPS極有可能參與了中年子鼠的認知功能加速衰退過程,甚至有可能對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.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R749.16

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