基于miR-132调控p250GAP信号途径探讨电针对睡眠剥夺大鼠突触可塑性和学习记忆作用的研究

Sleep deprivation (SD) leads to the decline of learning and memory ability, and synaptic plasticity is the neurobiological basis of learning and memory. MiR-132 can promote the development and differentiation of nerve cells, regulate the axon growth, branch and orientation of neurons, and affect synaptic plasticity and brain cognition, which may be a potential biological marker to improve the ability of learning and memory induced by SD. This project intends to use modified multi platform water environment method to prepare rat SD model, work around the working hypothesis that Electroacupuncture controls the p250GAP / Rac1 / Cdc42 signaling pathway by increasing the expression of miR-132, increasing the number and density of spinous processes, enhancing synaptic plasticity, improving learning and memory. Using Morris water maze to detect spatial learning and memory ability in rats, using Golgi staining to detect synaptic morphology plasticity, using electron microscopic to observe the synaptic ultrastructure in hippocampal CA1 region, using Patch clamp technique to detect LTP in hippocampal CA1 region, using Western blot and RT-PCR to detect the expression of miR-132, p250GAP, Rac1, Cdc42, So as to provide an experimental basis for acupuncture to improve the effective intervention target of SD-induced learning and memory ability, and to provide new ideas and methods for enriching the role of acupuncture in brain cognition.

睡眠剥夺（SD）导致学习记忆能力下降，神经元突触可塑性是学习记忆活动的神经生物学基础。MiR-132能促进神经细胞发育分化，调节神经元轴突生长、分支及定向，影响突触可塑性和脑认知，可能是针刺改善SD所致学习记忆能力下降的潜在生物学标志。本项目拟采用改良多平台水环境法制备大鼠SD模型，围绕“电针通过影响miR-132的表达调控p250GAP/Rac1/Cdc42信号通路，增加树棘突数量和密度，增强神经突触可塑性，改善学习记忆能力”的工作假说，运用水迷宫检测大鼠空间学习记忆能力，高尔基染色检测突触形态结构可塑性，电镜观察海马CA1区突触超微结构，膜片钳技术检测海马CA1区LTP，Western blot和RT-PCR检测miR-132、p250GAP、Rac1、Cdc42表达，从而为针灸改善SD所致学习记忆能力下降的有效干预靶点提供实验依据，为丰富针灸在脑认知领域中的作用提供新的思路和方法。

睡眠剥夺（SD）导致学习记忆能力下降，神经元突触可塑性是学习记忆活动的神经生物学基础。MiR-132能促进神经细胞发育分化，调节神经元轴突生长、分支及定向，影响突触可塑性和脑认知，可能是针刺改善SD所致学习记忆能力下降的潜在生物学标志。本研究采用电针干预睡眠剥夺大鼠，研究针刺对睡眠剥夺后大鼠海马神经元突触可塑性和学习记忆的调节作用，从 miR-132 入手，利用 miR-132 的激动剂和拮抗剂，来明确针刺对睡眠剥夺大鼠认知功能的提高是否是通过影响miR-132 的表达，从而调控 p250GAP 及其下游蛋白的可能机制。将60只SPF级雄性SD大鼠随机分为6组：空白组、模型组、模型+电针组、模型+假电针组、miR-132-agomir电针组、miR-132-antagomir电针组。给予相应干预后取材，分别采用PCR荧光技术检测各组大鼠海马miR-132、P250GAP表达水平；Western blot检测P250GAP、Rac1和Cdc42的蛋白表达量；免疫组化检测海马区p250GAP、Rac1、Cdc42 表达，高尔基染色检测大鼠海马CA1区树突棘数量，透射电镜观察海马 CAI 区突触的超微结构，膜片钳技术检测海马CA1区LTP。结果显示，电针可缩短睡眠剥夺大鼠morriss水迷宫逃避潜伏期，增加穿越平台次数和目标象限停留时间；增加睡眠剥夺大鼠海马miR-132基因蛋白表达，降低p250GAP基因蛋白表达，增加Rac1和Cdc42蛋白表达，增加海马树棘突数量，修复因睡眠剥夺导致的LTP下降；同时运用miR-132-agomir、miR-132-antagomir可证实电针是通过miR-132/p250GAP/Rac1/Cdc42通路来发挥调节突触可塑性的作用，为针灸在改善睡眠剥夺所致学习记忆能力下降的有效干预靶点提供实验依据，为丰富针灸在脑认知领域中的作用提供新的思路和方法。