miR-182和miR200b—Fbxw11和Rac1—Wnt信号通路在未成熟新生大鼠缺氧缺血性脑损伤时的作用

The mechanisms of preterm brain injury has been increasingly paid more attention.Our previous studies showed the expression of some miRNA was significantly downregulated in hypoxic-ischemic brain tissue ,found two significant differential expression miR-182 and miR-200b by analyzing hypoxic-ischemic brain tissue with miRNA chip. Based on the findings, we hypothesized that the two miRNA involved in Wnt signal passway through Fbxw11 and Rac1 gene. However, the effection on preterm brain injury and the mechanism remains unclear.We intend to bulit the hypoxic-ischemic animal model , transfect the full length and reverse sequence of miR-200b and miR-182 to brain ,develop over expression and low expression,observe the effection of the two miRNA on Fbxw11 and Rac1 gene, Wnt signal passway , oligodendroglia of different development stage ,injury and repair of myelin sheath and axon from the level of RNA and protein; on the shape and number of axons ,myelin sheath from the level of electron microscope; on the recognition ,memory and movement of animal from the point of neuroethology. The study findings will help to find new targets of rehabilitative treatment after preterm brain injury and provide experiment basis to these.

早产儿脑损伤的发生机制日益受到关注。我们前期的研究发现，缺氧缺血大鼠脑组织中一些miRNA表达明显下调，利用芯片技术对缺氧缺血后脑组织进行分析，筛选出差异表达明显的miR-182和miR-200b。根据预测结果，此两种miRNA可能通过Fbxw11和Rac1基因参与Wnt信号通路，但其对早产儿脑损伤结局的影响及具体机制还不清楚。因此，拟利用离体脑片和在体动物的缺氧缺血模型，将miR-200b和miR-182全长及反向序列转染到脑组织中造成过表达和低表达，分别进行：RNA和蛋白水平分析此两种miRNA对Fbxw11和Rac1基因、Wnt信号通路、不同发育阶段少突胶质细胞、髓鞘及轴突损伤、修复的影响；从电镜水平分析此两种miRNA对于轴突、髓鞘形态及数量的影响；从神经行为学角度分析它们对于动物的认知、记忆及运动等方面的影响。从而为早产儿脑损伤后脑功能的康复治疗寻找新的治疗靶点，并提供实验依据。

建立新生大鼠在体缺氧缺血脑损伤和离体脑片培养氧糖剥夺模型，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；第二，在细胞水平采用实时荧光定量PCR进行miR-200b转染的评价并检测Rac-1等基因的表达；第三、蛋白水平通过Western blot技术，检测β-catenin、β-trcp、GSK-3β、Rac-1以及MAPK、Rock2等的表达量变化，从而检验miR-200b是否通过调控Wnt信号通路来介导少突胶质细胞、髓鞘以及轴突等的功能变化进而影响缺氧缺血脑损伤的最终修复。最后，通过建立7日龄缺氧来对传统缺氧缺血脑损伤模型进行优化和改良，并与传统的缺氧缺血模型进行多个指标的比较。希望通过以上的研究，从一个新的角度研究缺氧缺血脑损伤以及修复的相关机制并为日后用于临床治疗提供可能的新的靶点。