本文關(guān)鍵詞： 脂聯(lián)素 三轉(zhuǎn)基因小鼠 學(xué)習(xí)記憶 突觸可塑性 炎癥　出處：《山西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討脂聯(lián)素(Adiponectin,APN)預(yù)處理能否有效改善9月齡APP/PS1/tau三轉(zhuǎn)基因阿爾茨海默病(3xTg-AD)模型小鼠的新物體識(shí)別、工作記憶和空間學(xué)習(xí)記憶能力損傷,緩解其焦慮情緒,逆轉(zhuǎn)其在體海馬突觸可塑性傷害,影響其海馬內(nèi)促炎因子NF-κB,IL-1β、TNF-α及抗炎因子IL-10的表達(dá)水平。從而證實(shí)脂聯(lián)素在AD病程中是否能發(fā)揮神經(jīng)保護(hù)作用,并探討其機(jī)制是否與減輕神經(jīng)炎性反應(yīng)有關(guān)。方法:實(shí)驗(yàn)選用9月齡雄性3xTg-AD小鼠和其背景鼠C57BL/6J小鼠,隨機(jī)分為四組,每組8只,分別為:野生對(duì)照組(WT+Saline)、野生給藥組(WT+APN)、三轉(zhuǎn)模型組(3xTg+Saline)和三轉(zhuǎn)給藥組(3xTg+APN)。連續(xù)10天經(jīng)側(cè)腦室分別注射0.1μg/4μl的脂聯(lián)素或等體積的生理鹽水,然后開始行為學(xué)實(shí)驗(yàn),在實(shí)驗(yàn)期間持續(xù)給藥直至斷頭取海馬組織。行為學(xué)實(shí)驗(yàn)分為四部分依次進(jìn)行:(1)曠場(chǎng)實(shí)驗(yàn):在安靜環(huán)境下,把小鼠放入曠場(chǎng)中央自由探索5 min,記錄小鼠運(yùn)動(dòng)的總距離及其在中央?yún)^(qū)域或外周區(qū)域的活動(dòng)時(shí)間占總時(shí)間的百分比。(2)新物體識(shí)別實(shí)驗(yàn):曠場(chǎng)實(shí)驗(yàn)后第2天進(jìn)行。在熟悉實(shí)驗(yàn)階段,將兩個(gè)完全相同的物體放入曠場(chǎng),再將小鼠從兩物體對(duì)側(cè)壁的中點(diǎn)貼壁放入,讓其自由探索10 min。小鼠休息5 h后,進(jìn)行測(cè)試實(shí)驗(yàn),將其中一個(gè)物體換為材質(zhì)相同、顏色形狀不同的新物體,讓小鼠再自由探索10 min。分析指標(biāo)包括物體識(shí)別指數(shù)和辨別指數(shù),物體識(shí)別指數(shù)=探索新物體時(shí)間或探索舊物體時(shí)間/(探索新物體時(shí)間+探索舊物體時(shí)間)×100%,辨別指數(shù)=(探索新物體的時(shí)間-探索舊物體的時(shí)間)/(探索新物體時(shí)間+探索舊物體時(shí)間)×100%。(3)Y迷宮自發(fā)交替實(shí)驗(yàn):將小鼠放入Y迷宮的三臂交匯區(qū)自由活動(dòng)8 min,記錄小鼠進(jìn)入各臂的總次數(shù)和順序,并計(jì)算其自發(fā)交替正確率。(4)Morris水迷宮實(shí)驗(yàn):首先進(jìn)行5天的定位航行實(shí)驗(yàn),每天將小鼠隨機(jī)放入4個(gè)象限。記錄其游泳軌跡、游泳速度和逃避潛伏期(找到水下平臺(tái)所用時(shí)間)。第6天上午進(jìn)行空間探索實(shí)驗(yàn),記錄60 s內(nèi)小鼠的運(yùn)動(dòng)軌跡及在目標(biāo)象限的活動(dòng)時(shí)間;下午進(jìn)行可視平臺(tái)實(shí)驗(yàn),記錄小鼠找到平臺(tái)的時(shí)間和游泳速度。行為學(xué)實(shí)驗(yàn)結(jié)束后,進(jìn)行在體海馬場(chǎng)電位記錄實(shí)驗(yàn)。將小鼠麻醉后固定在腦立體定位儀上,定位其海馬CA1區(qū)并將綁定的刺激電極和記錄電極插入。首先記錄30 min場(chǎng)興奮性突觸后電位(field excitatory postsynaptic potentials,f EPSPs),然后給予配對(duì)刺激記錄雙脈沖易化(paired-pulse facilitation,PPF),最后通過高頻刺激(high-frequency stimulation,HFS)誘導(dǎo)長(zhǎng)時(shí)程增強(qiáng)(long term potentiation,LTP)并記錄60 min。通過比較HFS前后各組f EPSPs的斜率來反映AD相關(guān)基因突變和脂聯(lián)素預(yù)處理是否影響突觸可塑性;通過比較各組的PPF值,來確定是否有突觸前神經(jīng)遞質(zhì)釋放的改變。在體LTP實(shí)驗(yàn)后,將小鼠斷頭取腦,通過Western blot實(shí)驗(yàn)檢測(cè)各組小鼠海馬內(nèi)促炎因子NF-κB、IL-1β、TNF-α和抗炎因子IL-10的表達(dá)量。結(jié)果:(1)脂聯(lián)素可以明顯改善9月齡3xTg-AD小鼠的識(shí)別記憶、工作記憶和空間學(xué)習(xí)記憶能力損傷,并緩解其焦慮情緒。在曠場(chǎng)實(shí)驗(yàn)中,與WT+Saline組相比,3xTg+Saline組小鼠在中央?yún)^(qū)域活動(dòng)的時(shí)間百分比明顯降低(P0.001),而在外周區(qū)域活動(dòng)的時(shí)間百分比明顯升高(P0.001),表明3xTg小鼠出現(xiàn)焦慮情緒;給予APN后,3xTg小鼠在中央?yún)^(qū)域活動(dòng)的時(shí)間增加,而在外周活動(dòng)的時(shí)間減少(P0.01),其焦慮情緒得到緩解。在新物體識(shí)別實(shí)驗(yàn)中,除3xTg+Saline組小鼠的新舊物體識(shí)別指數(shù)之間沒有統(tǒng)計(jì)學(xué)差異外,其余各組小鼠的新物體識(shí)別指數(shù)明均顯高于舊物體識(shí)別指數(shù)(P0.05)。同時(shí),3xTg+Saline組小鼠的辨別指數(shù)明顯低于WT+Saline組(P0.01),APN預(yù)處理后提高了3xTg小鼠的辨別指數(shù)(P0.01),表明APN能夠有效改善3xTg小鼠的新物體識(shí)別記憶損傷。在Y迷宮實(shí)驗(yàn)中,各組小鼠的總進(jìn)臂次數(shù)沒有統(tǒng)計(jì)學(xué)差異,但3xTg+Saline組小鼠的自發(fā)交替正確率明顯低于WT+Saline組和3xTg+APN小鼠(P0.01),提示脂聯(lián)素能夠改善3xTg小鼠的工作記憶損傷。在Morris水迷宮定位航行實(shí)驗(yàn)中,各組小鼠的逃避潛伏期均隨訓(xùn)練天數(shù)增加而明顯縮短。在第2-5天,3xTg+Saline組小鼠找到平臺(tái)所需時(shí)間明顯長(zhǎng)于WT+Saline組(P0.05);在第3-5天,3xTg+APN組小鼠的逃避潛伏期明顯短于3xTg+Saline組小鼠(P0.05)。在空間探索實(shí)驗(yàn)中,3xTg+Saline組小鼠在目標(biāo)象限的活動(dòng)時(shí)間百分比明顯少于其余三組(P0.001),且各組小鼠在6天中的游泳速度和到達(dá)可視平臺(tái)的時(shí)間均無統(tǒng)計(jì)學(xué)差異(P0.05),表明脂聯(lián)素預(yù)處理改善了3xTg小鼠空間學(xué)習(xí)記憶能力損傷。(2)脂聯(lián)素逆轉(zhuǎn)9月齡3xTg-AD小鼠的海馬突觸可塑性傷害。各組小鼠在HFS前30 min所記錄的f EPSPs斜率和配對(duì)脈沖刺激所引起的PPF比值均沒有統(tǒng)計(jì)學(xué)差異(P0.05)。當(dāng)給予HFS后,在四組小鼠均成功誘導(dǎo)出LTP。但在HFS后20 min開始,3xTg+Saline組小鼠f EPSPs的斜率明顯降低,在HFS后30 min和60 min,3xTg+Saline組小鼠f EPSPs的斜率都明顯低于WT+Saline組和3xTg+APN組小鼠(P0.05)。以上結(jié)果表明,AD相關(guān)基因突變和脂聯(lián)素預(yù)處理均未影響基礎(chǔ)突觸傳遞和突觸前神經(jīng)遞質(zhì)釋放,但脂聯(lián)素預(yù)處理可以有效逆轉(zhuǎn)3xTg小鼠的海馬突觸可塑性損傷。(3)脂聯(lián)素減輕了3xTg-AD小鼠海馬內(nèi)的神經(jīng)炎性反應(yīng)。Western blot實(shí)驗(yàn)結(jié)果顯示:與WT+Saline組相比,3xTg+Saline組小鼠海馬內(nèi)促炎因子NF-κB、IL-1β和TNF-α的表達(dá)量明顯升高(P0.01),而抗炎因子IL-10的表達(dá)量卻明顯降低(P0.001)。給予APN后,3xTg-AD小鼠海馬內(nèi)NF-κB、IL-1β和TNF-α的表達(dá)量有所下降(P0.05),而IL-10的表達(dá)量有所升高(P0.01)。結(jié)論:9月齡3xTg-AD小鼠的識(shí)別記憶、短期工作記憶和長(zhǎng)期空間學(xué)習(xí)記憶能力明顯損傷,并出現(xiàn)焦慮情緒。同時(shí),3xTg-AD小鼠還表現(xiàn)出在體海馬LTP的抑制和海馬內(nèi)神經(jīng)炎性反應(yīng)。給予脂聯(lián)素后,能夠改善3xTg-AD小鼠的學(xué)習(xí)記憶能力損傷、緩解其焦慮情緒和逆轉(zhuǎn)海馬LTP抑制,并且脂聯(lián)素可能是通過減少促炎因子表達(dá)和增加抗炎因子分泌,減輕腦內(nèi)神經(jīng)炎性反應(yīng),在AD病程中發(fā)揮神經(jīng)保護(hù)作用的。
[Abstract]:Objective: To investigate the effect of adiponectin (Adiponectin, APN) pretreatment can effectively improve the September age three APP/PS1/tau transgenic Alzheimer's disease (3xTg-AD) mouse model of new object recognition, working memory and spatial learning and memory damage, alleviate their anxiety to reverse the in vivo hippocampal synaptic plasticity in the hippocampus damage effects of proinflammatory cytokines NF- K B, IL-1 beta, the expression level of TNF- alpha and anti-inflammatory factor IL-10. To verify whether adiponectin in the course of AD can exert neuroprotective effects, and to explore its mechanism is associated with reduced neuroinflammatory response. Methods: twenty September old male 3xTg-AD mice and its background in C57BL/6J mice were randomly divided into four groups, each group 8, respectively: control group (WT+Saline), wild wild drug group (WT+APN), three (3xTg+Saline) and model group (3xTg+APN group) to three. 10 consecutive days were injected into lateral ventricle of 0.1 g/4 L The volume of saline or adiponectin, and behavioral experiments, during the experiment continuous administration until decapitated in hippocampus. The behavior experiment is divided into four parts: (1) in the open field test: in a quiet environment, the mice in the open field of central free exploration of 5 min, the total distance of movement of mice record and in the central region or peripheral regions of the percentage of the time in total time. (2) a new object recognition experiment: in the open field second days after the experiment. With the experimental stage, the two same objects in the open field, and then the mice from the two object point to the side wall of the wall let the freedom to explore into 10 min. mice rest after 5 h test, will be one of the objects for the same material, the new color of objects of different shapes, so the mice were then free to explore the 10 min. indicators including index index and object recognition to identify objects To explore a new object recognition index = time or time / explore old objects (Exploring the novel object exploration of old time + time object) * 100%, index of discrimination (= time exploring the novel object exploration of old objects (time) / time to explore new objects and explore the old object time) * 100%. (3) Y spontaneous maze alternate experiment: the mice were placed in the three arm maze of Y free intersection area of 8 min, and the total number of mice in each arm of the order of record, and calculate the correct rate of spontaneous alternation. (4) Morris water maze test: first locate 5 days of sailing experiment, mice with daily machine into the 4 quadrants. The swimming trajectory, swimming speed and escape latency (the time to find the platform under water). The morning of the sixth day to carry out space exploration experiment, recorded within 60 s mice and trajectories in the target quadrant time; the afternoon visual experimental platform, the platform to find records of mice And swimming speed behavior. After the experiment, the sea horse potential recording experiment. The mice were anesthetized on the stereotaxic apparatus, the location of the CA1 region of the hippocampus and the stimulating and recording electrodes inserted into the binding. First recorded in 30 min field excitatory postsynaptic potential (field excitatory postsynaptic potentials. F EPSPs), and then given the paired stimuli recorded paired pulse facilitation (paired-pulse facilitation, PPF), and finally by high frequency stimulation (high-frequency stimulation, HFS) induced long-term potentiation (long term, potentiation, LTP) and recorded 60 min. by comparing the slope of HFS f before and after EPSPs to reflect the AD gene mutation and adiponectin pretreatment whether the treatment effect of synaptic plasticity; through the comparison of the PPF value, to determine whether the presynaptic neurotransmitter release in the body. The change of LTP after the experiment, the mice brains were removed by W. Detection of estern blot mice hippocampus in experimental inflammatory factor NF- kappa B, IL-1 beta, TNF- alpha and the expression of anti-inflammatory factor IL-10. Results: (1) adiponectin can significantly improve the September age 3xTg-AD mice recognition memory, working memory and spatial learning and memory deficits, and alleviate their anxiety in the open field test. Compared with the WT+Saline group, 3xTg+Saline group, mice in the central area of activity time was decreased (P0.001), and the percentage of time in the peripheral areas of activity increased significantly (P0.001), showed that 3xTg mice had anxiety; for APN, the time in the central areas of activity of 3xTg mice increased in the peripheral the less time (P0.01), the anxiety eased. In the new object recognition experiments, in addition to no statistically significant difference between the old and the new object recognition index in 3xTg+Saline group, the new object recognition refers to the remaining mice The numbers were significantly higher than that of the old object recognition index (P0.05). At the same time, the 3xTg+Saline group discrimination index was significantly lower than in group WT+Saline (P0.01), APN after pretreatment improves the discrimination index of 3xTg mice (P0.01), show that APN can effectively improve the new object recognition memory in 3xTg mice in Y maze injury., no statistically significant differences between the total number of mice into the arm, but the spontaneous alternation of mice in 3xTg+Saline group was significantly lower than that of WT+Saline group and the correct rate of 3xTg+APN mice (P0.01), suggesting that adiponectin can improve working memory impairment in 3xTg mice. In Morris water maze navigation experiment, mice escape latency was increased significantly with the number of training days shortened. On day 2-5, 3xTg+Saline group of mice to find the platform required time was longer than that of group WT+Saline (P0.05); in the first 3-5 days, 3xTg+APN group mice escape latency was significantly shorter in the 3xTg+Saline group Rat (P0.05). In the space exploration experiment, 3xTg+Saline group in the target quadrant time percentage was significantly less than the other three groups (P0.001), and the swimming speed in 6 days in mice and reached the visual platform of the time were not statistically significant (P0.05), showed that adiponectin pretreatment improved the space 3xTg learning and memory ability of mice damage. (2) injury of hippocampal synaptic plasticity of adiponectin reversed in September aged 3xTg-AD mice. PPF mice in the ratio of 30 before HFS min recorded f EPSPs slope and paired pulse stimulation had no significant difference (P0.05). When given after HFS, in four groups of mice were successfully but after HFS induced LTP. 20 min, f EPSPs slope 3xTg+Saline group were significantly decreased after HFS, 30 min and 60 min, the slope of F EPSPs in 3xTg+Saline group were significantly lower than those of group WT+Saline and group 3xTg+APN mice (P0.05). The above results The result showed that the AD gene mutation and adiponectin pretreatment did not affect basal synaptic transmission and presynaptic neurotransmitter release, but adiponectin pretreatment can effectively reverse the 3xTg mice hippocampal synaptic plasticity damage. (3) adiponectin reduced 3xTg-AD mouse hippocampus in neuritic reaction of.Western blot results showed that compared with the WT+Saline group 3xTg+Saline group of proinflammatory cytokines in mice hippocampus NF- kappa B, expression of IL-1 beta and TNF- alpha were significantly increased (P0.01), whereas the expression of anti-inflammatory factor IL-10 was significantly lower (P0.001). After administration of APN, 3xTg-AD in NF- mice hippocampus kappa B, expression of IL-1 beta and TNF- alpha declined (P0.05), whereas the expression of IL-10 increased (P0.01). Conclusion: September 3xTg-AD mice at the age of recognition memory, short-term working memory and long-term spatial learning and memory ability was damaged and anxiety. At the same time, but also the performance of 3xTg-AD mice A neuroinflammatory response in the hippocampus and inhibition of hippocampal LTP. Given adiponectin, the ability of learning and memory damage can improve 3xTg-AD mice, alleviate their anxiety and reverse the inhibition of hippocampal LTP, and adiponectin may be reduced by proinflammatory cytokine expression and increased inflammatory factor secretion, reduce inflammatory reaction in brain in AD in the course of neuroprotection.

【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

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本文編號(hào)：1451612