Netrin-1与KIF1A在蛛网膜下腔出血后白质损伤中的作用机制研究

White matter injury after subarachnoid hemorrhage (SAH) is an important factor affecting the prognosis of SAH patients. Our preliminary experimental results showed that Netrin-1and KIF1A were probably involved in the pathological process of SAH-induced white matter injury. Thus, we propose the hypothesis: KIF1A mediates the secretion of Netrin-1. The expression of Netrin-1and KIF1A was decreased after SAH. In absent of Netrin-1, the dependent receptors of Netrin-1 promoted neuronal apoptosis and axonal demyelination, and finally white matter injury. To test the hypothesis, we firstly try to discover the effect of SAH on the expression of Netrin-1and KIF1A in the white matter of rats. Then, to choose the key clot component regulating the expression of Netrin-1and KIF1A, different clot components were separately used to treat cultured neurons. And the key clot component was injected into the brain to test its effects. Third, gene transfection, RNAi and transgenic mice are used to regulate the target gene expression, and then to evaluate the role of Netrin-1and KIF1A on SAH-induced white matter injury. And finally, by double immunofluorescence and co-immunoprecipitation, we demonstrate the mechanisms underlying the effect of Netrin-1and KIF1A on SAH-induced white matter injury. The longitudinal studies from three levels of cell, tissue and in vivo, horizontal researches from three levels of molecular biology, neuroimaging and molecular biology in this study will provide us new ideas and theoretical basis to improve the prognosis of SAH.

蛛网膜下腔出血（SAH）后发生白质损伤。我们的预实验结果提示：Netrin-1/KIF1A可能参与SAH后白质损伤。为此，我们提出假说：KIF1A介导Netrin-1分泌，SAH后二者表达下降；Netrin-1缺失导致其依赖型受体启动神经元凋亡和轴突脱髓鞘，促进白质损伤。为了验证该假说，我们拟利用大鼠SAH模型，应用western blot等检测SAH后白质中Netrin-1/KIF1A的水平；运用血肿产物处理神经元，筛选调控Netrin-1/KIF1A表达的关键血肿产物，并进行活体验证；应用基因调控和转基因，结合磁共振成像等技术，评估调控Netrin-1/KIF1A表达对白质损伤的影响；利用CO-IP等技术，阐述Netrin-1/KIF1A调控白质损伤的作用机制。本研究纵向从细胞、组织和活体三个层面，横向从分子生物学，神经影像和行为学三个层面开展研究，将为改善 SAH 预后提供新思路。

Netrin-1称为神经导向因子，其神经保护及修复作用在多种模型中也已被证实，然而netrin-1在脑出血（intracerebral hemorrhage ICH）中的研究尚未见报道。在本研究中，我们发现ICH后脑组织中netrin-1的表达量呈上升趋势，48小时达到高峰；ICH后netrin-1的受体DCC、UNC5H2也呈显著上升趋势。ELISA试剂盒测定脑脊液中netrin-1的含量，我们发现脑出血后netrin-1的分泌量呈上升趋势，72小时达高峰，较ICH后netrin-1的表达变化趋势延后大约24小时。COIP结果提示ICH后，与netrin-1结合的受体DCC和UNC5H2较sham组有所增加，但结合的受体明显低于ICH后实际表达总量。通过蛋白质印迹及免疫荧光法发现ICH后驱动蛋白KIF1A的表达量呈显著下降趋势。Netrin-1干预后，我们观察到netrin-1组与PBS组相比，TUNEL、FJB阳性率及caspase-3表达量显著降低，所以侧脑室注射netrin-1后可显著抑制脑出血后的神经元死亡。KIF1A干预后，我们发现over-KIF1A组与Vehicle组相比，TUNEL、FJB阳性率及caspase-3表达量显著降低。用ELISA测定脑脊液及细胞培养液中netrin-1含量，我们发现上调KIF1A后netrin-1含量明显上升；相反，下调KIFIA后其含量下降。值得注意的是干预KIF1A后脑组织中netrin-1的表达量并无明显变化，仅分泌量受到影响。综上我们发现，Netrin-1可阻断其依赖性性受体DCC和UNC5H2介导的凋亡通路发挥神经保护作用。驱动蛋白KIF1A作为分子马达，参与netrin-1分泌过程中的胞内运输，影响netrin-1的胞外释放，从而在实验性ICH后的EBI中发挥着抑制细胞死亡、神经保护的作用。