本文選題：睡眠剝奪　切入點(diǎn)：記憶　出處：《第二軍醫(yī)大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：大量的研究已經(jīng)證實(shí)睡眠剝奪與認(rèn)知損害密切相關(guān)，但潛在的機(jī)制尚不明確。已有的研究發(fā)現(xiàn)細(xì)胞型朊蛋白（cellular prion protein，PrP~C）在睡眠調(diào)節(jié)和記憶過(guò)程中發(fā)揮重要作用，并且它和阿爾茨海默病有多方面的聯(lián)系，而阿爾茨海默病突出的臨床特征是記憶衰退。是否PrP~C在睡眠剝奪導(dǎo)致的認(rèn)知損害中發(fā)揮作用值得研究。 首先，我們把雄性成年Sprague-Dawley大鼠隨機(jī)分為3組，分別為：籠養(yǎng)對(duì)照組（cage control group，CC組），，水槽對(duì)照組（tank control group，TC組）即環(huán)境對(duì)照組，睡眠剝奪組（sleep deprivation group，SD組）。用改良多平臺(tái)睡眠剝奪方法給予大鼠連續(xù)72h的睡眠剝奪。用Morris水迷宮測(cè)試評(píng)價(jià)海馬依賴的空間記憶能力。完成睡眠剝奪及水迷宮測(cè)試后，用western blot技術(shù)檢測(cè)大鼠不同腦區(qū)PrP~C表達(dá)水平。結(jié)果顯示大鼠睡眠剝奪后海馬依賴的空間記憶受損，PrP~C的表達(dá)在海馬選擇性下調(diào)。 CREB是已知的海馬依賴的學(xué)習(xí)、記憶存儲(chǔ)和突觸可塑性的關(guān)鍵調(diào)節(jié)因子。已有的研究顯示大鼠海馬p-CREB的水平在學(xué)習(xí)后增加，在睡眠剝奪后減低，提示CREB磷酸化在睡眠剝奪導(dǎo)致的認(rèn)知損害中發(fā)揮重要作用。PrP~C參與調(diào)節(jié)多個(gè)信號(hào)轉(zhuǎn)導(dǎo)通路的活性，但是否PrP~C調(diào)節(jié)海馬CREB磷酸化尚未見(jiàn)報(bào)道。為了初步探討睡眠剝奪后記憶損害的相關(guān)信號(hào)通路，我們按照上述動(dòng)物分組，進(jìn)一步用westernblot技術(shù)和免疫熒光組織化學(xué)技術(shù)研究了72-h睡眠剝奪對(duì)大鼠不同腦區(qū)p-CREB表達(dá)的影響。然后在原代培養(yǎng)的Sprague-Dawley大鼠海馬神經(jīng)元中，用慢病毒介導(dǎo)的RNA干擾技術(shù)沉默PrP~C，用western blot技術(shù)檢測(cè)p-CREB表達(dá)的變化。我們發(fā)現(xiàn)72-h睡眠剝奪導(dǎo)致大鼠海馬p-CREB的表達(dá)選擇性下調(diào)，而總CREB的表達(dá)未受影響，這與已有的研究報(bào)道相符合。給予原代培養(yǎng)的海馬神經(jīng)元針對(duì)PrP~C的RNA干擾后7天，p-CREB的表達(dá)水平明下調(diào)。 已有的研究高度提示成體海馬新生的神經(jīng)元參與學(xué)習(xí)和記憶。而軸突的形成和生長(zhǎng)是神經(jīng)元分化中首要的形態(tài)學(xué)改變，這使得神經(jīng)元之間能夠進(jìn)行信息傳遞。學(xué)習(xí)和記憶等這些高級(jí)神經(jīng)活動(dòng)更是依賴于神經(jīng)元之間信息的傳遞。已有的研究提示PrP~C在神經(jīng)元軸突延伸中發(fā)揮作用。為了驗(yàn)證低水平的PrP~C對(duì)海馬神經(jīng)元軸突延伸的影響，我們?cè)趤?lái)自新生Sprague-Dawley大鼠的原代培養(yǎng)的海馬神經(jīng)元中，仍舊用慢病毒介導(dǎo)的RNA干擾技術(shù)沉默PrP~C，然后用免疫熒光細(xì)胞化學(xué)技術(shù)觀察海馬神經(jīng)元軸突生長(zhǎng)情況。結(jié)果顯示RNA干擾后7天，海馬神經(jīng)元軸突的生長(zhǎng)明顯受到抑制。 這些發(fā)現(xiàn)提示，PrP~C很可能在睡眠剝奪造成的海馬依賴的空間記憶損害過(guò)程中發(fā)揮作用，而CREB信號(hào)通路可能參與其中。軸突生長(zhǎng)抑制則可能是直接導(dǎo)致認(rèn)知功能損害的結(jié)構(gòu)基礎(chǔ)。這些結(jié)果也許能夠部分解釋睡眠剝奪影響認(rèn)知功能的機(jī)制�；谶@些發(fā)現(xiàn)，PrP~C，CREB，軸突延伸有可能成為干預(yù)睡眠剝奪后認(rèn)知障礙的潛在的靶點(diǎn)。
[Abstract]:A large number of studies have confirmed that sleep deprivation is closely related to cognitive impairment, but the underlying mechanism is not clear. It has been found that cellular prion protein (prion) plays an important role in sleep regulation and memory. And it has multiple links to Alzheimer's disease, which is characterized by memory decline. Whether PrP~C plays a role in cognitive impairment caused by sleep deprivation is worth investigating. Firstly, male adult Sprague-Dawley rats were randomly divided into three groups: cage control group CC group and tank control group TC group. Rats were given sleep deprivation for 72 hours by modified multi-platform sleep deprivation method. The spatial memory ability of hippocampal dependence was evaluated by Morris water maze test. After sleep deprivation and water maze test were completed, the sleep deprivation and water maze test were completed. Western blot technique was used to detect the expression of PrP~C in different brain regions of rats. The results showed that the expression of PrPnC was selectively down-regulated in the hippocampus of rats with hippocampal dependent spatial memory impairment after sleep deprivation. CREB is known as a key regulator of hippocampal dependent learning, memory storage and synaptic plasticity. It is suggested that CREB phosphorylation plays an important role in the cognitive impairment induced by sleep deprivation. PrPnC is involved in regulating the activity of multiple signal transduction pathways. However, whether PrP~C regulates the phosphorylation of CREB in the hippocampus has not been reported. In order to explore the signal pathway related to memory impairment after sleep deprivation, we grouped the animals according to the above mentioned animals. The effects of 72-h sleep deprivation on the expression of p-CREB in different brain regions of rats were further studied by westernblot technique and immunofluorescence histochemical technique. Then, in primary cultured hippocampal neurons of Sprague-Dawley rats, The expression of p-CREB was silenced by lentivirus mediated RNA interference technique, and the expression of p-CREB was detected by western blot. We found that 72-h sleep deprivation induced selective down-regulation of p-CREB expression in hippocampus of rats, but the expression of total CREB was not affected. The expression of p-CREB in primary cultured hippocampal neurons was down-regulated 7 days after RNA interference with PrP~C. Previous studies have highly suggested that adult hippocampal neonate neurons participate in learning and memory. The formation and growth of axons are the primary morphological changes in neuronal differentiation. These advanced neural activities, such as learning and memory, depend on the transmission of information between neurons. Previous studies have suggested that PrP~C plays a role in neuronal axon extension. The effects of low level PrP~C on axonal extension of hippocampal neurons were tested. We were in primary cultured hippocampal neurons from newborn Sprague-Dawley rats, The growth of axons of hippocampal neurons was observed by immunofluorescence cytochemical technique. The results showed that the axonal growth of hippocampal neurons was significantly inhibited 7 days after RNA interference. These findings suggest that PrPnC may play a role in the hippocampus dependent spatial memory impairment caused by sleep deprivation. The inhibition of axon growth may be the structural basis of cognitive impairment. These results may partly explain the mechanism of sleep deprivation affecting cognitive function. The axon extension may be a potential target for cognitive impairment after sleep deprivation.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16

【共引文獻(xiàn)】

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