miR-200b通过Rac1和Slit2调节未成熟新生大鼠缺氧缺血脑损伤髓鞘修复和轴突导向的机制

The treatment of brain damage in preterm infant has no breakthrough because of the repairment mechanism still unclear. Our previous chip studies found the expression of miR-200b was significantly decreased in hypoxic-ischemic premature rat brain,however,the expression of Rac1 and Slit2 were significantly increased when knockdown miR-200b in cultured brain slice. The effection of miR-200b on hypoxia/ischemia brain damage(HIBD) and regulation mechanism remain unknown,therefore, we plan to transfect miR-200b and reverse sequence to hypoxic-ischemic premature rat brain, analyze the influence on hypoxia/ischemia through neurobehavioral evaluation, the effection of miR-200b on oligodendrocyte proliferation and axon regeneration by tracer technique, morphologic variations by electron microscope, mitochondria activity from metabolic level.Then detect the regulation of miR-200b on Rac1 and Slit2 by luciferase reporter system in vitro, observe the proliferation, migration of oligodendrocyte and axon growth, try to explain the role of miR-200b on myelin sheath and axon, confirm whether miR-200b regulating myelin sheath repairment and axon guidance through Rac1 and Slit2 in premature rat with HIBD, provide scientific basis for treatment.

早产儿脑损伤的修复机制尚不明确，致使其治疗无突破性进展。我们前期芯片研究发现，缺氧缺血未成熟大鼠脑组织miR-200b表达明显下调，在培养脑片中敲低miR-200b，Rac1和Slit2表达均明显提高，但miR-200b对缺氧缺血性脑损伤结局的影响及其机制尚不明确。本研究拟将miR-200b及反向序列转染至未成熟大鼠缺氧缺血脑组织，通过神经行为评估等了解其对缺氧缺血结局的影响；利用示踪技术分析miR-200b对少突胶质细胞增殖及轴突再生的影响,电镜检测形态学变化，能量代谢水平检测线粒体活性。然后在离体水平用荧光素酶报告系统检验miR-200b对Rac1及Slit2的靶向调节作用，分析少突胶质细胞增殖、迁移情况及轴突生长的变化,进而揭示其对髓鞘以及轴突影响的具体机制；以检验"mir-200b是否通过Rac1和Slit2调节未成熟新生大鼠缺氧缺血脑损伤髓鞘修复和轴突导向"，为治疗提供实验依据。

建立了未成熟新生大鼠在体缺氧缺血脑损伤模型，通过纳米聚合载体转染miR-200b agomir和miR-200b antagomir将miR-200b进行上调/下调，进行如下研究：首先，利用水迷宫和转棒实验等进行大鼠神经行为学评价，探讨miR-200b对缺氧缺血脑损伤过程中学习记忆和运动协调等能力的影响，通过对线粒体膜电位的检测探讨了miR-200b对缺氧缺血脑损伤大鼠线粒体活性的影响；第二，通过Western blot方法检测了Wnt信号通路各重要节点蛋白Rac-1、β-TrCP、GSK-3β、β-catenin的表达情况，分析了miR-200b对Wnt信号通路的影响，通过实时荧光定量PCR及Western blot方法分别在核酸水平和蛋白水平分析了miR-200b对缺氧缺血大鼠轴突导向因子Slit2、Robo1的影响；第三、进行大鼠神经元细胞原代培养与鉴定，并通过细胞免疫荧光技术分析miR-200b对离体培养神经元骨架及轴突的影响。通过以上的研究，从一个新的角度研究了缺氧缺血脑损伤以及修复的相关机制并为今后用于临床治疗提供可能的新的靶点。