血流切应力影响斑马鱼血管发育的力学生物学机制研究

The vascular development is decided by genetic factors to relative high extent. However recent research indicated that hemodynamics factors such as shear stress are essential factor in embryonic vascular development. Unfortunately, the mechanical molecular mechanism is poorly understood. To confirm the mechanobiological mechanism which blood flow shear stress regulates vascular development, we hypothesized that the effect of shear stress on vascular development is achieved via the regulation of klf2a and nos2b expression though ERK5 signal pathway, which is caused by the change of cytoskeleton. In this research, we used the Flk1: GFP×Gata1：DsRed transgenic zebrafish as an animal model to investigate the involvement of shear stress in vascular development by the modulators of blood flow and gene knockdown approaches, then the relationship between blood flow shear stress and vascular development was analyzed by laser scanning confocal microscopy and some softwares. Moreover, we will study the molecular mechanism of klf2a and nos2b involved in the process of vascular development using the approaches of molecular and cellular biology. Further more,based on the change of cytoskeleton, we will investigate the mechanobiological mechanism which vascular development mediated by the induction of klf2 and nos2b gene expression though ERK5 signal pathway which was caused by the change of cytoskeleton by blood flow shear stress. This study will establish and develop a method to bring the mechanical factor into the study of developmental biology, and provide a new way for further understanding and clarifying the mechanism of mechanobiology which blood flow shear stress regulates vascular development.

血管发育在很大程度上由遗传控制，而近年研究发现血流切应力调节也是血管发育的必要因素，但其相关的力学生物学分子机制尚未阐明。为明确血流切应力调节血管发育的力学生物学机制，本项目提出如下假设：血流切应力参与调节血管发育过程，且是通过改变血管内皮细胞的细胞骨架状态、介导ERK5信号通路进而调节klf2a和enos基因表达来实现的。本项目以双转基因斑马鱼(flk1: GFP×gata1: DsRed)为研究对象，通过血流调节剂和基因敲降改变血流、激光共聚焦拍摄和软件分析，量化血流切应力对血管发育的影响；运用分子生物学方法明确klf2a和nos2b基因参与血流切应力调节血管生成过程；探讨力学因素诱导的细胞骨架改变介导ERK5信号通路调控klf2a和nos2b影响血管发育过程的机制。本研究拟建立一套将力学因素引入发育生物学研究的方法，为深入理解和阐明血流切应力调节血管发育的力学生物学机制提供新依据。

血流动力学和血管发育以及重塑密切相关，但是其机制并未清楚。本项目以斑马鱼为模型，通过激光共聚焦显微镜结合活体成像技术、基因敲降、基因编辑以及分子细胞生物学等技术系统的研究了斑马鱼尾部静脉血管丛（CVP）发育的整个过程，并且定量分析血流动力学对CVP血管新生和CV形成中的作用和机制。我们发现斑马鱼尾部静脉血管发育分为三个清晰的阶段：CVP血管新生阶段、CV形成阶段、CVP血管消退阶段。改变血流动力学扰乱斑马鱼CVP血管新生。定量分析表明血流切应力的变化和CVP发育的变化呈线性关系。机制上血流动力学通过激活ERK5-klf2a-nos2b信号通路促进CVP血管新生。与此同时，我们发现CV形成主要包括两个方式：血管融合和血管修剪。随后通过激光共聚焦结合斑马鱼活体成像技术发现：当分叉的两根血管中血流速度相同时，分叉的两根血管进行融合，最终形成CV。当分叉的两根血管中血流速度不一样的时候，血流速度大的血管将稳定保留下来，最终形成CV，血流速度小的血管发生内皮细胞重排，被修剪掉。机制上，我们发现血流动力学-klf6a-tagln2信号通过通过促进内皮细胞迁移和细胞骨架聚合调控斑马鱼CV形成。本研究已初步建立一套将力学因素引入发育生物学研究的方法和模型，为深入理解和阐明血流切应力调节血管发育的力学生物学机制提供了新依据。