力学敏感YAP通路介导流体剪切力调控血脑屏障的力学生物学机制探索

Cerebrovascular diseases are one of the most serious diseases that threaten human health. The haemodynamics play a central role in maintaining the structure and function of blood-brain barrier (BBB) in cerebral vessels. However, under the condition of normal physiology, ischemia hypoperfusion and hypertransfusion, the mechanisms that how fluid shear stress (FSS) protect, damage and destruct BBB are still unclear. The activation of YAP is closely related to the intercellular contact inhibition and tight junction. Our previous results indicated that, under the physiological condition, FSS upregulated the expression of YAP, while high FSS could induce the cytoskeleton rearrangement of rBMECs, increased the intercellular permeability, and downregulate the YAP level. Accordingly, we speculated that fluid shear stress regulated the structure and function by YAP pathway. Using novel flow chamber system and cerebral ischemia rat model, this project aims to examine the effects of different intensity of FSS on the structure and function of cerebrovascular endothelial cells and BBB. Additionally, the time-dependent expression and distribution of key proteins in the “Integrin/YAP/Rho GTPases/F-actin” signal pathway will be studied, and the mechanism of activated YAP in nucleus will be analyzed to further explore the mechanobiologic mechanisms that YAP induced FSS regulating BBB. The finding of this project would provide the suggestions and guides for the design and improvement of cerebrovascular interventional implants.

脑血管疾病是威胁人类健康的重大疾病，血流动力学对于维持血脑屏障（BBB）的结构和功能具有决定性作用。然而，在正常生理、缺血低灌注和搭桥术后高灌注下，脑血管中不同强度的剪切力对BBB的保护、损伤和破坏机制尚未明确。力学敏感蛋白YAP的活性与细胞间接触抑制和紧密连接密切相关。我们前期结果表明：生理水平的剪切力上调YAP的表达；而高剪切力诱导rBMECs通透性增加，YAP表达显著降低。由此推测：剪切力通过YAP通路调控BBB结构和功能。本项目拟通过新型流动腔系统及脑缺血大鼠模型，探讨流体剪切力对rBMECs紧密连接和通透性的影响，通过解析“Integrin-YAP-Rho GTPases-F-actin”信号轴上关键蛋白表达和分布的时序规律，以及YAP入核活化机理，明确在生理和病理条件下，YAP参与流体剪切力调控BBB的力学生物学机制。研究结果对脑血管介入材料的设计具有重要的指导意义。

脑血管疾病是威胁人类健康的重大疾病，血流动力学对于维持血脑屏障（BBB）的结构和功能具有决定性作用。严重的脑血管狭窄或闭塞患者需要血管搭桥手术进行治疗，重建闭塞脑血管的血供。由于缺血部位的病变血管长期暴露于低灌注和低剪切的应力环境，颅内搭桥段血管血流量的迅速增加引起流体剪切力（FSS）和灌注压急剧增大，破坏搭桥段血管末端血脑屏障，导致部分患者在术后出现严重的高灌注综合征。然而，在正常生理、缺血低灌注和搭桥术后高灌注下，脑血管中不同强度的剪切力对BBB的保护、损伤和破坏机制尚未明确。本项目通过优化设计的新型流动腔建立脑血管内皮共细胞培养模型，施加不同强度的FSS，实时观察BBB结构的变化；明确YAP入核活化调控细胞胞间紧密连接的分子机制；通过大鼠中动脉闭塞模型（MCAO）进行在体验证，明确FSS调控BBB的力学生物学机制。研究结果表明：（1）构建的体外模拟BBB的新型流动腔模型，其内部流体为充分发展的泊肃叶流。该模型可实时观测内皮细胞在力学刺激下的形态变化等，并实时监测细胞层通透性的变化。（2）正常生理条件下FSS有助于维持BBB结构的完整性，而低剪切或高剪切均会损伤或破坏BBB的结构；FSS对BBB的调控与紧密连接和粘附连接相关蛋白的表达和分布密切相关。（3）Integrin β1在细胞膜响应不同强度的力学信号，通过Rho GTPases实现对细胞骨架F-actin的调控，细胞骨架抑制剂显著抑制YAP入核活化，证实了FSS调控血脑屏障依赖于Integrin-Rho GTPases-F-actin-YAP信号通路。（4）构建大鼠MACO在体模型，验证了缺血再灌注前后BBB结构和功能的变化，证实力学敏感YAP通路介导FSS调控BBB。本项目的研究结果有助于从新的角度阐述血流动力学对BBB的保护和破坏机制，给国内外众多从事血管生物力学和心脑血管介入材料研究的科研工作者以新的启示。