Slit3/Robo4调控integrinα5β1介导的脉络膜血管新生"定向侵袭"机制研究

The mechanism of choroidal neovascularization invation was complicate and rarely reported. Our previous research revealed that integrinα5β1 can be singled out as one of the primary proteins required for CNV as a VEGF-independent pathway, which may explain why anti-VEGF monothreapy doesn't work in some cases. Recently, it was found that neuronal guidance signal Slit3/Robo4 pathway participated into regulation of endothelial cell migration and angiogenesis. So it suggested that Slit3/Robo4 pathway might play a key role in the regulation of integrinα5β1 pathway mediated CNV invasion. Therefore, activation/inhibition of Slit3/Robo4 pathway will be investigated by construction Robo4 gene overexpression and silence plasmid to explore whether Slit3/Robo4 is the upstream regulatory pathway involving in integrinα5β1-mediated CNV invastion.The effector of Robo4-integrionα5β1 binding on choroidal endothelial cell migration,invasion, tube formation will also be explored by overexpression/inhibition Robo4 gene. In conclusion, the critical molecular and its mechanism in CNV directional invasion will be investigated to test its therapeutic potential in CNV diseases in this research.

脉络膜新生血管（CNV）的"定向侵袭"机制复杂且少有报道。课题组前期研究发现integrinα5β1能独立于VEGF/VEGFR通路之外，单独或协同VEGF/VEGFR通路，诱导形成CNV侵袭，且不被VEGF抗体所抑制；新近发现Slit3/Robo4信号参与调控内皮细胞定向迁移，介导新生血管形成，提示Slit3/Robo4可能是integrinα5β1介导的CNV"定向侵袭"的上游调控信号之一。以此为基础，课题组拟在体内外CNV模型中，采用基因过表达/沉默技术,研究Slit3/Robo4信号对integrinα5β1通路表达影响，及对脉络膜血管内皮细胞迁移、侵袭、管腔形成的影响，及其探讨其参与CNV"定向侵袭"的分子机制，阐明Slit3/Robo4-integrinα5β1活化是CNV"定向侵袭"的重要调控机制之一；为我们进一步认识CNV形成机制，为提高抗VEGF治疗临床疗效提供理论依据。

课题组首先明确integrinα5基因在血管新生过程中的发挥的生物学功能，其次明确其在小鼠脉络膜新生血管中定位及相关信号通路分子的表达，在激光诱导脉络膜新生血管动物眼内，应用免疫荧光、共聚焦显微镜，观察CNV侵袭Bruch’s膜过程中 integrinα5表达，活性改变；构建integrinα5敲除质粒，转染原代脉络膜血管内皮细胞（CEC），integrinα5敲除CEC能够有效抑制细胞增殖、迁移、管腔形成；采用integrinα5联合抗VEGF药物，能够有效抑制新生血管形成、改善血管通透性；在此基础上，应用Real-Time PCR 、初步筛选可能的通路Slit/Robo4相关信号分子,发现Robo4可能是integrinα5的上游调控分子。本研究结果已经显示integrinα5在脉络膜新生血管形成中的重要作用，联合integrinα5拮抗剂与抗VEGF药物更加有效抑制实验性脉络膜新生血管，为临床今后联合用药抑制CNV提供了一定的理论依据。项目研究成果在Molecular pharmaceutics等期刊发表。