Olig2通过SHH信号通路调控早产儿缺氧缺血脑损伤神经修复的机制

Neuronal injury is one of important pathological mechanisms when neonatal hypoxic-ischemic brain damage occurs. In our previous study, we discovered that the number of surviving neurons increased after knock down the expression of oligodendrocyte leaneage transcription factor 2 (Olig2). While its effects on neuronal function and the detailed mechanism is still unclear. Bioinformatics analysis suggested that Olig2 may thus play a role by interacting with NKX2-2 etc.through SHH signaling pathway in the surviving neurons. In this study, we want to establish hypoxic-ischemic brain damage model and knock down Olig2 in vivo and in vitro，respectively. Firstly, knock down Olig2 in vivo, neurobehavioral evaluation is conducted through Morris water maze experiments and so on to judge the ultimate effect of Olig2 knock-down to brain injury repair; secondly, investigate the impact of Olig2 knock-down on neuronal proliferation , death and the impact on axon regeneration in vivo; thirdly, investigate the proteins of SHH signaling pathway which may be involved in these processes in vitro, furthermore, the agonists, inhibitors, and other means of intervention such as siRNA and overexpression are used to intervene key proteins in SHH signaling pathway, and then analyze its effects on neuronal function and quantity; Finally, we will use chromatin immunoprecipitation-sequencing to find out the genes regulated by Olig2. We hope this study can provide some new targets for future clinical treatment to neonatal hypoxic-ischemic brain damage.

新生儿缺氧缺血脑损伤时神经元损伤是重要的病理机制。我们前期发现，少突胶质细胞系转录因子2（Olig2）下调可使神经元存活增加，但是其对神经元功能的影响及相关机制尚不明确。生物信息学分析提示Olig2可能通过调控NKX2-2等进而影响SHH信号通路发挥作用。本研究拟建立缺氧缺血脑损伤Olig2敲低模型，从在体动物和离体神经干细胞两个层面进行研究，首先，在体通过水迷宫实验等神经行为学评价探讨Olig2敲低对脑损伤修复的最终影响；第二，在体研究Olig2敲低对神经元增殖、死亡及轴突再生的作用；第三，离体水平敲低Olig2，分析可能参与上述过程的SHH信号通路的变化，通过特异抑制剂、激活剂以及siRNA和过表达等手段干预SHH信号通路节点蛋白，分析其对神经元功能和数量的影响；最后，离体水平通过染色质免疫共沉淀-测序来研究Olig2调控的相关基因。为新生儿缺氧缺血脑损伤的临床治疗提供可能的分子靶标。

新生儿缺氧缺血脑损伤时神经元损伤是重要的病理机制。前期研究发现，少突胶质细胞系转录因子2（Olig2）下调可使神经元存活增加，本研究拟探讨下调Olig2对神经元功能的影响的具体机制。3日龄SD大鼠随机分成3组：正常对照组、RNAi对照组、Olig2 RNAi敲低组，通过水迷宫检测记忆能力、转棒分析检测运动行为能力、免疫组化和免疫荧光检测细胞增殖情况，通过实时定量PCR检测SHH信号通路相关基因的表达，通过Western blot检测凋亡相关基因，将SD大鼠分成两组，通过CHIP-seq进行分析。 缺氧缺血脑损伤发生后给予Olig2的敲低，发现大鼠的找到平台的潜伏期缩短，各组的潜伏期均随着时间推移而逐渐缩短，缺氧缺血脑损伤发生之后进行Olig2的敲低能够使记忆能力恢复到与正常对照组相当的程度，可见缺氧缺血脑损伤发生早期进行Olig2的敲低对于脑损伤具有一定的修复作用；Olig2敲低组的大鼠在棒时间是优于HI组的有统计学差异，其他时间点的检查结果没有统计学差异；免疫组化结果发现在胼胝体区，Olig2敲低组的细胞增殖的数量比缺氧缺血组多，在海马齿状回和海马CA1区的Olig2敲低组细胞增殖与缺氧缺血组相当，但是均少于正常对照组，在海马CA2区和LV区Olig2敲低组细胞增殖少于缺氧缺血组；免疫荧光结果提示在海马齿状回Olig2敲低组增殖的神经元少于缺氧缺血组；实时定量PCR的结果提示，Olig2敲低组的Smo表达较RNAi对照组有明显的降低，Gli1的表达也有类似趋势，Western blot结果提示Olig2敲低组的凋亡相关基因包括Cathepsin B和BECN1与缺氧缺血组相比均有降低的趋势。