睡眠剥夺对大鼠不同脑区PrP~C表达的影响及PrP~C在睡眠剥夺诱导的认知损害中可能的作用

发布时间：2018-03-13 10:27

本文选题：睡眠剥夺　切入点：记忆　出处：《第二军医大学》2012年博士论文　论文类型：学位论文

【摘要】：大量的研究已经证实睡眠剥夺与认知损害密切相关，但潜在的机制尚不明确。已有的研究发现细胞型朊蛋白（cellular prion protein，PrP~C）在睡眠调节和记忆过程中发挥重要作用，并且它和阿尔茨海默病有多方面的联系，而阿尔茨海默病突出的临床特征是记忆衰退。是否PrP~C在睡眠剥夺导致的认知损害中发挥作用值得研究。首先，我们把雄性成年Sprague-Dawley大鼠随机分为3组，分别为：笼养对照组（cage control group，CC组），，水槽对照组（tank control group，TC组）即环境对照组，睡眠剥夺组（sleep deprivation group，SD组）。用改良多平台睡眠剥夺方法给予大鼠连续72h的睡眠剥夺。用Morris水迷宫测试评价海马依赖的空间记忆能力。完成睡眠剥夺及水迷宫测试后，用western blot技术检测大鼠不同脑区PrP~C表达水平。结果显示大鼠睡眠剥夺后海马依赖的空间记忆受损，PrP~C的表达在海马选择性下调。 CREB是已知的海马依赖的学习、记忆存储和突触可塑性的关键调节因子。已有的研究显示大鼠海马p-CREB的水平在学习后增加，在睡眠剥夺后减低，提示CREB磷酸化在睡眠剥夺导致的认知损害中发挥重要作用。PrP~C参与调节多个信号转导通路的活性，但是否PrP~C调节海马CREB磷酸化尚未见报道。为了初步探讨睡眠剥夺后记忆损害的相关信号通路，我们按照上述动物分组，进一步用westernblot技术和免疫荧光组织化学技术研究了72-h睡眠剥夺对大鼠不同脑区p-CREB表达的影响。然后在原代培养的Sprague-Dawley大鼠海马神经元中，用慢病毒介导的RNA干扰技术沉默PrP~C，用western blot技术检测p-CREB表达的变化。我们发现72-h睡眠剥夺导致大鼠海马p-CREB的表达选择性下调，而总CREB的表达未受影响，这与已有的研究报道相符合。给予原代培养的海马神经元针对PrP~C的RNA干扰后7天，p-CREB的表达水平明下调。已有的研究高度提示成体海马新生的神经元参与学习和记忆。而轴突的形成和生长是神经元分化中首要的形态学改变，这使得神经元之间能够进行信息传递。学习和记忆等这些高级神经活动更是依赖于神经元之间信息的传递。已有的研究提示PrP~C在神经元轴突延伸中发挥作用。为了验证低水平的PrP~C对海马神经元轴突延伸的影响，我们在来自新生Sprague-Dawley大鼠的原代培养的海马神经元中，仍旧用慢病毒介导的RNA干扰技术沉默PrP~C，然后用免疫荧光细胞化学技术观察海马神经元轴突生长情况。结果显示RNA干扰后7天，海马神经元轴突的生长明显受到抑制。这些发现提示，PrP~C很可能在睡眠剥夺造成的海马依赖的空间记忆损害过程中发挥作用，而CREB信号通路可能参与其中。轴突生长抑制则可能是直接导致认知功能损害的结构基础。这些结果也许能够部分解释睡眠剥夺影响认知功能的机制。基于这些发现，PrP~C，CREB，轴突延伸有可能成为干预睡眠剥夺后认知障碍的潜在的靶点。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

【共引文献】