DCC受体及其信号通路在介导netrin-1蛋白诱导脑缺血后血管新生中的机制

Angiogenesis is very important for the rescue of brain injury after cerebral ischemia. The increase of exogenous Netrin-1(NT-1) can promote angiogenesis around the ischemic area and improve the recovery of neurological function, However, the mechanism under which is unclear. DCC receptor expressed in brain vascular endothelial cells. Moreover, the change of DCC expression in the brain after ischemic injury is consistent with the change of NT-1, which indicate that the NT-1/DCC signaling pathway plays a key role in the brain angiogenesis after ischemia. This project will use brain vascular endothelial cells oxygen-glucose deprivation model and mice brain ischemia model to test: ①whether NT-1 promotes the proliferation, migration and tuber formation of endothelial cells by the DCC dependent ERK1/2-eNOS signaling pathway in the oxygen-glucose deprivation model; ②whether the overexpression of NT-1 promotes local angiogenesis around the ischemic area by the DCC dependent ERK1/2-eNOS signaling pathway in the transient middle cerebral artery occlusion mice model. By using siRNA, specific antibodies and inhibitors etc., we will identify the NT-1/DCC signaling pathway and the downstream gene which mediated angiogenesis after cerebral ischemic injury and furthermore discover the mechanism of DCC-mediated angiogenesis. Different from neuroprotection, this subject will provide a new idea for the treatment of cerebral ischemia.

血管新生对脑缺血后的损伤修复至关重要。外源性增高轴突诱向因子Netrin-1（NT-1）能促进缺血周围区的血管新生，促进神经功能恢复。但其作用机制还不清楚。脑血管内皮细胞上表达DCC受体，且在缺血损伤后的脑组织中DCC表达量的动态变化与NT-1相一致，提示NT-1/DCC信号在促进脑内血管新生中起关键作用。因此推测NT-1可能通过DCC受体信号通路及下游基因促进脑缺血后的血管新生。本课题将通过siRNA、特异性抗体、抑制剂干扰等手段，利用脑血管内皮细胞糖氧剥夺和小鼠大脑中动脉缺血模型，研究：①NT-1是否通过DCC依赖的ERK1/2-eNOS信号通路促进糖氧剥夺时内皮细胞的增殖、迁移和形成管腔；②在短暂性脑缺血小鼠模型中，NT-1过表达是否通过DCC依赖的ERK1/2-eNOS信号通路促进局部血管新生。与单一的神经保护不同，本课题的研究结果将为脑缺血的治疗提供一个思路。

血管新生对脑缺血后的损伤修复至关重要。外源性增高轴突诱向因子Netrin-1（NT-1）能促进缺血周围区的血管新生，促进神经功能恢复。但其作用机制还不清楚。脑血管内皮细胞上表达DCC受体，且在缺血损伤后的脑组织中DCC表达量的动态变化与NT-1相一致，提示NT-1/DCC信号在促进脑内血管新生中起关键作用。因此推测NT-1可能通过DCC受体信号通路及下游基因促进脑缺血后的血管新生。我们在分离的小鼠脑微血管内皮细胞中通过免疫组化的方法证实在脑微血管内皮细胞上存在NT-1的DCC和UNC5H2受体。证实NT-1对脑微血管内皮细胞的增殖具有双重导向作用，低剂量具有促进增殖作用而随着剂量增加促进作用减弱，甚至表现出抑制作用，并由此确定了NT-1作用的最佳浓度。在加入DCC和UNC5H2受体的抗体后NT-1的促脑微血管内皮细胞增殖作用得到了抑制，表明DCC和UNC5H2受体均参与了NT-1促进的脑微血管内皮细胞增殖过程。NT-1的促脑微血管内皮细胞迁移作用在加入DCC和UNC5H2受体的抗体后仅UNC5H2受体抗体对NT-1的促脑微血管内皮细胞迁移起到了抑制，表明UNC5H2受体参与了NT-1促进脑微血管内皮细胞的迁移过程。推测NT-1对血管调控存在一个复杂的网络结构，这是我们今后研究的方向。