Phactr-1对创伤后脑血管内皮细胞及血脑屏障损伤修复作用及其分子机制研究

After traumatic brain injury (TBI), the recovery of neural function depends on not only the neuron self-repair, but also the structure and function integrity of the brain blood vessels and blood-brain barrier. Our preliminary study found that there was an increase on the expression of phosphatase and actin regulator (Phactr-1) after TBI in rats. Sometimes, this increase can promote brain vascular endothelial cell proliferation, and decrease cell apoptosis, but its impact on endothelial cells and blood brain barrier after TBI and the mechanisms remains to be further explored.We hypothesized that: Phactr-1 regulates the activity of PP1, inhibits apoptosis pathways, and reduces endothelial cell apoptosis after TBI. According to the endothelial cell stretch injury model and the mice cortex control injury model, this project will analyze the change of Phactr-1 expressions, firstly. Secondly, we will further construct the Phactr-1 siRNA and the lentiviral vectors for the open reading frame of Phactr-1, intracerebroventricular injection and lentiviral transfection were processed to modify expression of Phactr-1, and observe the impact on PP1 activity. This study will discuss the impact of these changes on the structure and function of brain endothelial cells and BBB after TBI, and explore the vascular protective effects and mechanism of Phactr-1 after TBI.

创伤性脑损伤（TBI）后神经功能修复不仅需要神经细胞自身修复，也有赖于脑血管和血脑屏障的结构和功能完整。课题组前期发现，TBI后大鼠脑组织磷酸酶 和肌动蛋白调节因子（Phactr-1）表达增高，并可促进脑血管内皮细胞的增殖、减少凋亡，但其对TBI后脑血管内皮细胞及血脑屏障的作用和机理尚待进 一步探讨。我们推测：Phactr-1通过调节PP1活性，抑制相关凋亡通路，减少内皮细胞凋亡，对TBI后BBB损伤发挥修复作用。为此，本项目拟通过 建立血管内皮细胞牵张损伤模型和小鼠皮层控制损伤模型，分析Phactr-1表达变化；进一步构建针对Phactr-1的siRNA及针对其开放阅读框架的慢病毒载体，通过慢病毒小鼠侧脑室注射和细胞转染，改变Phactr-1表达，并观察对PP1活性的影响，同时探讨上述变化对TBI后脑血管内皮细胞及 BBB结构和功能的影响，期望发现Phactr-1对TBI后的血管保护作用与机理。

创伤性脑损伤（TBI）后神经功能修复不仅需要神经细胞自身修复，也有赖于脑血管和血脑屏障的结构和功能完整。课题组前期发现，TBI后大鼠脑组织磷酸酶 和肌动蛋白调节因子（Phactr-1）表达增高，并可促进脑血管内皮细胞的增殖、减少凋亡。在该课题中，课题组建立了稳定小鼠控制性皮层损伤模型和bEnd.3细胞牵张损伤模型，验证了phactr-1在小鼠创伤性颅脑损伤以及脑血管内皮细胞牵张损伤中的重要作用。随着颅脑损伤后时间的延长，phactr-1的表达水平越来越高。在bEnd.3细胞牵张损伤模型中，发现过表达phactr-1可促进细胞增殖、迁移，减少凋亡，且凋亡相关因子表达水平较对照组明显降低。在小鼠皮层控制性损伤模型中，过表达phactr-1能显著减轻由损伤导致的血脑屏障破坏、脑水肿程度、损伤灶大小、细胞凋亡等，验证了在phactr-1过表达后的bEnd.3细胞凋亡水平、氧化应激相关蛋白表达降低，ZO-1蛋白、Occludin蛋白、抗凋亡蛋白表达增加，提示在颅脑创伤中phactr-1发挥作用很有可能是通过凋亡相关机制进行调节。以上实验结果可能为颅脑创伤治疗提供新策略。