發(fā)布時(shí)間：2018-01-06 21:16

  本文關(guān)鍵詞：缺鋅致大鼠學(xué)習(xí)記憶損傷的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制研究　出處：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 缺鋅 海馬 信號(hào)轉(zhuǎn)導(dǎo) 學(xué)習(xí)記憶 CREB

【摘要】： 目的:在成功制備缺鋅大鼠動(dòng)物模型的基礎(chǔ)上,觀察缺鋅對(duì)生長(zhǎng)大鼠認(rèn)知行為、突觸傳遞效能、抗氧化功能及海馬神經(jīng)細(xì)胞凋亡的影響,重點(diǎn)探討缺鋅對(duì)生長(zhǎng)期大鼠海馬cAMP/PKA和MEK/ERK信號(hào)通路中幾種重要信號(hào)分子表達(dá)的影響,以期闡明缺鋅致認(rèn)知功能損傷的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。方法:(1)缺鋅大鼠模型的建立。健康雄性Wistar大鼠96只,體重60g～80g,隨機(jī)分為三組:足鋅組(ZA)、缺鋅組(ZD)、對(duì)喂組(PF),其中ZD組喂飼缺鋅飼料(1.5mg/kg),ZA組和PF組喂飼足鋅飼料(30mg/kg)。以當(dāng)日Z(yǔ)D組攝食量作為次日PF組的給料量。動(dòng)物單籠喂養(yǎng),自由飲用去離子水。實(shí)驗(yàn)期一個(gè)月。(2)鋅營(yíng)養(yǎng)狀況評(píng)價(jià)。采用火焰原子吸收法測(cè)定大鼠血漿鋅和海馬鋅含量,TSQ熒光法檢測(cè)海馬CA3區(qū)鋅的分布和含量,連續(xù)監(jiān)測(cè)法測(cè)定血漿堿性磷酸酶(ALP)活性。(3)學(xué)習(xí)記憶能力檢測(cè)。通過(guò)被動(dòng)回避實(shí)驗(yàn)和Y-迷宮實(shí)驗(yàn)觀察缺鋅對(duì)實(shí)驗(yàn)大鼠學(xué)習(xí)記憶能力的影響。(4)LTP誘導(dǎo)實(shí)驗(yàn)。通過(guò)誘導(dǎo)大鼠海馬齒狀回LTP觀察缺鋅對(duì)大鼠海馬突觸傳遞效能的影響。(5)生化指標(biāo)的檢測(cè)。采用堿性羥胺比色法分別測(cè)定全血、海馬及皮層乙酰膽堿(Ach)的含量;熒光分光光度法測(cè)定海馬、皮層單胺類神經(jīng)遞質(zhì)多巴胺(DA)、去甲腎上腺素(NE)、5-羥色胺(5-HT)的含量。采用硫代巴比妥酸法測(cè)定大鼠血清、海馬和皮層丙二醛(MDA)含量;采用相關(guān)試劑盒分別檢測(cè)大鼠血清、海馬和皮層超氧化物歧化酶(SOD)活性、總抗氧化力(T-AOC)。以流式細(xì)胞術(shù)檢測(cè)大鼠海馬神經(jīng)細(xì)胞凋亡率。(6)信號(hào)通路中信號(hào)分子的檢測(cè)。采用放射免疫分析法(RIA)測(cè)定大鼠血漿、海馬和皮層組織cAMP含量;應(yīng)用PepTag?檢測(cè)系統(tǒng)測(cè)定海馬和皮層PKA活性。分別應(yīng)用Western blot和RT-PCR法檢測(cè)大鼠海馬和皮層p-MEK、p-ERK1/2、p-CREB和BDNF蛋白的表達(dá)水平以及CREB mRNA、BDNF mRNA的轉(zhuǎn)錄水平。結(jié)果:(1)實(shí)驗(yàn)期間,ZD組大鼠精神萎靡,活動(dòng)減少,生長(zhǎng)遲緩,并逐漸出現(xiàn)明顯的脫毛、嘴角炎和趾間炎等缺鋅癥狀。(2)與ZA組和PF組比較,ZD組大鼠體重、攝食量、血漿鋅含量、海馬CA3區(qū)鋅含量、ALP活力均明顯降低(P0.05)。(3)與ZA組和PF組相比,ZD組大鼠的避暗潛伏期明顯縮短(P0.05),在Y迷宮測(cè)試中的錯(cuò)誤反應(yīng)次數(shù)顯著增加(P0.05)。(4)三組大鼠均誘導(dǎo)出LTP;但ZD組大鼠在高頻刺激后,各個(gè)時(shí)間點(diǎn)的群峰電位(PS)增長(zhǎng)幅度較ZA組和PF組顯著降低(P0.05)。(5)與ZA組和PF組相比,ZD組大鼠全血Ach含量顯著升高(P0.05),海馬Ach含量有下降趨勢(shì),但無(wú)顯著性差異;ZD組大鼠皮層Ach含量與ZA組相比顯著降低(P0.05),但與PF組比較無(wú)顯著性差異;ZD組腦組織NE、5-HT、DA等單胺類神經(jīng)遞質(zhì)含量較ZA組和PF組不同程度升高(P0.05)。(6)與ZA組和PF組相比,ZD組大鼠血清、海馬和皮層MDA含量顯著增加(P0.05), SOD活性及T-AOC降低(P0.05);并且ZD組大鼠海馬神經(jīng)細(xì)胞凋亡率顯著升高(P0.05)。(7)與ZA組比較,ZD組和PF組大鼠海馬cAMP含量顯著升高(P0.05),ZD組較PF組有升高趨勢(shì);而ZD組大鼠血漿和皮層cAMP含量顯著高于ZA組和PF組(P0.05),ZA組與PF組間無(wú)差異;ZD組大鼠海馬和皮層PKA活性較ZA組和PF組均顯著降低(P0.05),PF組又顯著低于ZA組(P0.05)。(8)ZD組海馬和皮層p-MEK、p-ERK1/2蛋白表達(dá)水平較ZA組和PF組均明顯下調(diào),而ZA組和PF組之間沒(méi)有顯著性差異。(9)與ZA組和PF組相比, ZD組海馬和皮層p-CREB、BDNF蛋白表達(dá)水平及CREB mRNA、BDNF mRNA水平均明顯下調(diào),而ZA組和PF組之間也分別有不同程度的改變。結(jié)論:(1)成功建立了缺鋅大鼠動(dòng)物模型。(2)鋅缺乏可使大鼠學(xué)習(xí)記憶功能受損。(3)鋅缺乏可導(dǎo)致大鼠海馬腦區(qū)突觸傳遞效能降低,膽堿能及單胺類神經(jīng)遞質(zhì)含量異常改變,抗氧化防御系統(tǒng)功能降低,海馬神經(jīng)元凋亡率增高,進(jìn)而在行為上表現(xiàn)出學(xué)習(xí)記憶功能降低。(4)鋅缺乏可導(dǎo)致大鼠海馬和皮層cAMP-PKA及MEK-ERK信號(hào)通路中主要信號(hào)分子發(fā)生異常改變,使CREB磷酸化水平下降,BDNF蛋白表達(dá)水平下降,從而抑制LTP的形成并損傷學(xué)習(xí)記憶,這可能是缺鋅致學(xué)習(xí)記憶功能損傷的重要分子機(jī)制。
[Abstract]:Objective: to prepare basic zinc deficient rats in animal models were successfully on the observation of zinc deficiency on the growth of cognitive behavior in rats, synaptic transmission efficiency, the effects of antioxidant function and neuronal apoptosis in the hippocampus, focusing on the effects of zinc deficiency on the expression of several important signal molecules in rat hippocampal cAMP/PKA and MEK/ERK signaling pathway in the growth period, in order to clarify the signal transduction mechanism of zinc deficiency induced cognitive impairment. Methods: (1) establishment of zinc deficient rat model. 96 healthy male Wistar rats, weight 60g ~ 80g, were randomly divided into three groups: foot group (ZA), zinc deficiency group (ZD), pair fed group (PF), ZD group fed with zinc deficient diet (1.5mg/kg), ZA group and PF group were fed the zinc adequate feed (30mg/kg). On the same day as the food intake of group ZD group PF the next day to feed the animal. Single cage feeding, drinking deionized water. The experimental period of one month. (2) evaluate the zinc nutritional status was determined by large. Flame atomic absorption spectrometry Plasma zinc and zinc content in hippocampus, the content and distribution of detection in hippocampal CA3 region of zinc TSQ fluorescence method for determination of plasma alkaline phosphatase (ALP) activity of continuous monitoring method. (3) the ability of learning and memory testing. Through the passive avoidance test and Y- maze test to evaluate the influence of zinc deficiency on rat memory ability of learning (4.) induced by LTP experiment. Through the induction of LTP in the rat dentate gyrus examined effects on hippocampal synaptic transmission efficacy in rats. (5) biochemical detection. Blood were measured by colorimetric method of alkaline hydroxylamine, hippocampus and cerebral cortex acetylcholine (Ach) content of hippocampus; determination of fluorescence spectrophotometry, cortex monoamine the neurotransmitter dopamine (DA), norepinephrine (NE), 5- serotonin (5-HT). The contents of serum were measured by thiobarbituric acid method, the hippocampus and the cortex malondialdehyde (MDA) content in serum of rats were detected; the related kit, hippocampus and skin The layer of superoxide dismutase (SOD) activity, total antioxidant capacity (T-AOC). To detect the apoptosis rate of hippocampal nerve cells by flow cytometry. (6) the detection signals in signalingpathways. Radioimmunoassay (RIA) determination of rat plasma, the content of cAMP in hippocampus and cortex tissue; application PepTag? PKA activity in hippocampus and cortex were detected respectively by the Western system. Blot and RT-PCR were detected in the cortex and hippocampus of p-MEK, p-ERK1/2, p-CREB and BDNF protein expression levels of CREB and mRNA, the transcriptional level of BDNF mRNA. Results: (1) during the experiment, ZD rats, listlessness, decreased activity, growth slowly and gradually apparent hair removal, and toe inflammation mouth inflammation and other symptoms of zinc deficiency. (2) compared with ZA group and PF group, ZD group, body weight, food intake, plasma zinc content, zinc content of CA3 in hippocampus, the activity of ALP was significantly decreased (P0.05) compared with ZA (3). Group and PF group, ZD group 榧犵殑閬挎殫娼滀紡鏈熸槑鏄劇緝鐭，

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