PTEN泛素化调控NF-κB信号通路参与颅脑创伤后微血栓形成的作用及机制研究

The microthrombosis formation after traumatic brain injury (TBI) is an important factor leading to secondary ischemic brain injury. NF-κB activation can break the dynamic balance between coagulation and fibrinolysis to promote microthrombosis formation. We found that PTEN was involved in the activation of NF-κB after cerebral ischemia, and the inhibition of PTEN could reduce the apoptosis of neurons and the permeability of blood brain barrier after TBI. However, the impact of PTEN on the TBI induced microthrombosis has not been elucidated. We speculate that the ubiquitination of PTEN plays an important role in microthrombosis formation after TBI via the regulation of NF-κB signal pathway. To verify this hypothesis, we will observe the PTEN ubiquitination and NF-κB activation after TBI in the brain microvascular endothelial cells (BMVEC) stretch injury model and mice cortex controlled injury model. Furthermore, the overexpression or RNA interference of E3 ligase CHIP via lentivirus transfection was applied to regulate PTEN ubiquitination and expression, in order to evaluate the impact of PTEN ubiquitin modification on NF-κB activity, the function of BMVEC and the microcirculation after TBI. Thus, this program is expected to clarify the role and mechanism of PTEN ubiquitination in TBI induced microthrombosis formation and provide experimental basis for the treatment of ischemic brain injury after TBI.

创伤性脑损伤(TBI)后的微血栓形成是导致继发性脑缺血的重要因素。NF-κB活化能够打破纤凝平衡，促进微血栓形成。课题组前期发现，PTEN参与了脑缺血后NF-κB的活化；抑制PTEN可以减少TBI后神经元凋亡，降低血脑屏障通透性，但其对TBI后微血栓形成的作用和机理尚未阐明。我们推测，颅脑创伤后，PTEN的泛素化修饰能够通过调控NF-κB信号通路，参与挫伤灶及周边组织微血栓形成。为此，本项目拟通过建立脑微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PTEN泛素化和NF-κB的活化情况；进一步构建泛素连接酶CHIP过表达或siRNA的慢病毒载体，通过细胞转染和侧脑室注射，观察PTEN泛素化修饰对NF-κB活性的影响，同时探讨上述变化对TBI后脑微血管内皮细胞功能及损伤区微循环的影响，进而阐明其在创伤后脑组织微血栓形成的作用及机制，为寻找TBI后缺血性脑损伤防治方法提供实验依据。

创伤性脑损伤（TBI）可以打破凝血和纤溶系统之间的动态平衡，促进微血栓形成，这是导致继发性缺血性脑损伤的重要因素。PTEN参与脑缺血和TBI后AKT的激活，抑制PTEN则有助于TBI后的神经保护。然而，PTEN对TBI诱导的微血栓形成的影响尚未阐明。我们推测PTEN泛素化通过调节AKT信号通路在TBI后的微血栓形成中发挥重要作用。在本研究中，我们通过过表达和敲减泛素连接酶CHIP来调节PTEN蛋白变化，并进一步调节TBI后小鼠脑微血管内皮细胞（BMVEC）和组织中的AKT信号通路，分别测定体外细胞增殖、迁移、侵袭能力和细胞凋亡情况，同时检测TBI后PTEN泛素化引起的促凝血和细胞凋亡分子的变化，以及体内TBI后微血管密度和微血栓形成。体外实验结果表明，CHIP过表达促进BMVECs细胞增殖，迁移和侵袭，下调细胞凋亡。进一步的体内实验证明，CHIP过表达通过抑制TBI后细胞凋亡和凝血过程，从而减少TBI后微血栓和增加微血管密度。我们的研究结果还表明CHIP的抗细胞凋亡和凝血作用与TBI中的PTEN / AKT信号通路密切相关。综上所述，本研究认为PTEN泛素化可以通过促进细胞增殖，抑制细胞凋亡和机体凝血过程，从而抑制TBI后的微血栓形成。