早期心理应激对小鼠成年后学习记忆及其海马区基因表达模式的影响

Early-life psychological stress can cause anxiety, pessimism and the decrease of cognitive ability in adult individuals. The stress-induced gene expression pattern can remain until the adult stage. In this study, the pure psychological stress model (terrified sound stress model) would be applied to new-born male mouse for fourteen days, and Morris Water Maze would be employed to test their learning and memory abilities during their adulthood. The difference of hippocampal proteomic expression in the stressed adult mouse would be characterized using bidirectional electrophoreses, and further identified using mass spectrometry and bioinformatic techniques. The identified proteins would be then analyzed for their tissue localization and expression levels using immunohistochemical and Western-blot techniques, which would illuminate the effects of early-life psychological stress on the gene expression pattern in the hippocampus of adult mouse. The differentially expressed genes, for which mRNA and proteins display identical expression patterns, would be detected for promoter methylathion, and thus the mechanism for how the stress can affect the epigenetic pattern would be preliminarily illustrated. Our study would lay a solid theoretical foundation for investigating the effects of early-life stress on individuals during their adulthood. The highlights of the present study include: (1) the use of pure psychological stress model for investigating the changes in the learning and memory abilities of stressed mouse after their transition into their adulthood; and (2) the use of proteomic techniques for analyzing the effects of early-life stress on the gene expression pattern during their adulthood.

早期心理应激会引起个体成年后情绪焦虑、消极以及学习记忆等认知能力的降低。提示应激诱导建立的基因表达模式可以保留至成年期。本研究采用单纯心理应激模型(恐惧声音心理应激)应激新生小鼠14天；水迷宫检测其成年后的学习记忆能力；利用双向电泳检测应激小鼠成年后海马蛋白组表达差异，并对其进行质谱鉴定和生物信息学分析；采用免疫组化及Western-blot对鉴定出的蛋白进行组织定位及表达水平的验证，利用Real-Time PCR检测其mRNA表达水平，阐明早期心理应激对小鼠成年后海马基因表达模式的影响；对mRNA与蛋白表达变化一致的差异表达基因进行启动子的甲基化检测，初步探索应激影响表观遗传模式建立的机制。为婴幼儿心理应激对成年后的影响提供理论依据。研究特色与创新点：１.采用了单纯心理应激模型研究应激小鼠成年后学习记忆能力的变化；2.应用蛋白组学分析早期心理应激对应激个体成年后基因表达模式的影响。

基于本课题组建立的恐惧声音-心理应激模型，对新生鼠进行2周的恐惧声音心理应激及母子隔离的应激，2周后水迷宫检测发现恐惧声音心理应激组及母子隔离组与对照比相比，空间学习记忆能力显著降低，待其成年后，水迷宫检测发现，与对照组相比，成年后的恐惧声音组和母子隔离组大鼠的空间学习记忆能力显著降低，表明幼年期应激造成的空间学习记忆能力的损伤可以维持至成年期，应激鼠与对照组的海马蛋白表达差异分析表明，早期心理应激可以引起大鼠海马区蛋白表达的显著改变，且部分蛋白如微管蛋白TUBB、肌球蛋白MYOSIN-4、肌动蛋白Actin及血清白蛋白Serum Albumin, 甘油酸磷酸脱氢酶Glyceraldehyde-3-phosphate dehydrogenase等蛋白的显著改变可以持续到成年。表明幼年的心理应激的确会导致成年后海马基因表达模式的改变。此外，课题组采用该恐惧声音-心理应激模型对青春期SD大鼠进行应激后的海马区进行蛋白组学研究，恐惧声音-心理应激对大鼠的海马蛋白表达产生显著的改变，应激鼠海马有52个蛋白表达明显下调，35个蛋白显著上调，并对其中的6个差异最显著的蛋白进行了质谱鉴定，结果表明蛋白HSP71、TIM、GFAP在应激组大鼠海马区显著下调，而蛋白TUBB、LDH-A、CKB在应激后表达显著升高。因此，可以总结为，恐惧声音心理应激皆可以引起早期及青春期的大鼠海马区蛋白表达的改变，对早期海马蛋白表达的影响部分可以维持到成年，推测其中表观遗传学机制起作用。应激鼠海马DNA的甲基化测序正在进行中，期待此部分有更深层的机制研究结果。