紧密连接蛋白Claudin-5在斑马鱼胚胎血管发生中的作用机制研究

As a critical structural and functional component of tight junction, Claudin-5 functions to seal paracellular space between the endothelial cells, such as in blood-brain barrier, blood-retina barrier. During the development of zebrafish central nervous system, Claudin-5a helps to establish a neuroepithelial-ventricular barrier, which is required for brain ventricular expansion. Our recent study also reveals that zebrafish Claudin-5a and Claudin-5b are essential for zebrafish vasculogenesis. Loss of both Claudins prevents the separation of arterial and venous cells without affecting their differentiation. Based on our preliminary results, we propose that it is the adhesion function of Claudin-5 which affects the cell motility and cell-cell adhesion behavior during the separation of endothelial cells. This non-canonical function is separated from Claudin's sealing function in tight junction belt. In this study, we will combine the traditional methods of developmental biology and modern biophysical methods to analyze the new developmental roles of Claudin-5s in zebrafish vasculogenesis,including investigating the genetic relationships between Claudin-5s and Vegf or Ephrin on the vasculogenesis singaling pathway. We will also analyze the effect of Claudin-5s on the endothelial cell adhesion function and migration behavior. This study will reveal the non-canonical roles of Claudin-5s in vivo for the first time. It will also improve the understanding of the signaling pathways which regulate vascular endothelial cell differentiation during artery and vein formation. These may have an important impact on understanding the mechanism of mammalian vasculature formation and regeneration.

Claudin-5作为重要的紧密连接蛋白，在血脑屏障、血视网膜屏障等组织屏障中具有维持细胞间通透性的功能。我们最新研究发现,Claudin-5在斑马鱼胚胎血管发生中也起着重要的发育生物学作用，其缺失影响动静脉血管内皮细胞的分离，最终形成单一融合的畸形血管。结合前期实验基础，我们提出了"Claudin-5所介导的细胞间黏附作用决定了胚胎发育早期动静脉血管内皮细胞的分离与迁移过程"这一假说。本课题将检测Claudin-5与血管发生信号通路中Vegf,Ephrin等的相互关系，同时结合生物物理学分析方法，重点研究Claudin-5对血管内皮细胞间黏附作用力及细胞迁移行为的影响，探讨Claudin-5在斑马鱼血管形成中的作用机制，证实课题提出的设想。本研究将首次在模式动物体内揭示Claudin新的发育学功能，进一步完善胚胎血管发育中的信号分子调控网络，从而为研究人类血管发育和再生过程奠定坚实基础。

哺乳动物Claudin-5 (Cldn5)作为重要的紧密连接蛋白，在血管内皮细胞中特异性表达，对维持血脑屏障、血视网膜屏障等组织屏障的通透性具有重要的功能。本项目的前期研究中，我们发现斑马鱼Cldn5的缺失影响动静脉血管内皮细胞的分离，并最终形成单一融合的畸形血管的。在此项目的研究过程中，我们提出并通过体、内外实验验证了" Cldn5所介导的血管内皮细胞间黏附作用决定了胚胎发育早期动静脉血管内皮细胞的分离与迁移过程"这一假说。结合体内转基因斑马鱼、cldn5a, cldn5b基因敲低、敲除斑马鱼品系，体外细胞模型等研究揭示了：1.内皮细胞Cldn5的缺失影响了斑马鱼胚胎早期动、静脉血管发生；2. Cldn5缺失不会导致血管发生信号通路中Vegf, EfnB-2a等的改变；3.结合生物物理学分析方法，利用和改进原子力显微镜技术等检测表明Cldn5具有血管内皮细胞间的黏附作用并决定血管细胞的迁移行为。该项目在执行期内较为完整地解决了项目提出的科学问题，证实了课题当初提出的科学设想。本研究首次在细胞水平和模式动物体内揭示了Claudin蛋白新的发育生物学功能，进一步完善胚胎血管发育中的信号分子调控网络，从而为研究人类血管发育和再生过程奠定坚实基础。