CCM下游信号通路基因在血管发育缺陷及脑中风发病中的分子遗传机制研究

Stroke is a major cause of death and disability in China. Although conventional risk factors such as lipid profiles, smoking, and hypertension accounted for a considerable part of the stroke events, most subjects with these risk factors do not develop stroke during their lifetime, suggesting that other factors are also involved. Endothelial cell-cell adhesion is important for proper blood vessel formation, maintaining the integrity of the vasculature. Weakened endothelial junctions are implicated in the etiology of stroke, especially intracerebral hemorrhagic stroke (ICH). ..The zebrafish is a useful model organism for analysis of human disease genes and their pathophysiology. Our preliminary work identified a zebrafish mutant fn40a of beta-Pix gene, a guanine nucleotide exchange factor, which exhibited embryonic cerebral hemorrhage with abnormal vascular structure. Beta-Pix, Rap1b, and Pak2a potentially function in a signaling pathway downstream from CCM gene products, and there is a combinational interaction between these genes predispose to hemorrhagic stroke. These data provide an important clue for exploring the molecular mechanisms underlying human stroke. ..In this study, we will systematically assess the role of CCM pathway genes (Beta-Pix, Rap1b, and Pak2a) in stroke etiology by using different integrated approaches which include basic cell biology, studies in animal model (zebrafish), and clinic studies. First, we will use zebrafish mutant to analyze the gene functions for vascular integrity. Secondly, we will investigate the association between the genetic variations in the CCM pathway and stroke risk in a well-established case-control study and further confirm these results in a prospective study. Finally, we will perform additional experiments to further examine how these risk genetic variations function for proper endothelial cell-cell junction formation in human endothelial cells and in zebrafish. Our research will help for understanding the high variability in appearance and onset of the stroke, and provide useful new molecular targets for the diagnosis and treatment of stroke.

脑中风发病率高，致死、致残率高，负担日益沉重。前期工作利用斑马鱼筛选发现调控血管发育的重要基因beta-Pix。 Beta-Pix与Rap1b, Pak2a, CCMs处于同一信号通路，基因突变可引起斑马鱼脑出血，为揭示人类脑中风的分子遗传机理提供了重要线索。本课题采用分子遗传、模式生物和人群队列研究相结合的策略，拟解决关键问题1：利用遗传修饰斑马鱼的优势，筛选血管发生异常的突变体，分析beta-Pix等基因对血管功能的影响；问题2：在病例-对照和前瞻性随访队列中系统检测上述基因变异是否能够预测脑中风发病和复发风险；问题3：在细胞水平，分析基因突变对人血管内皮细胞迁移、增殖等作用；利用定点突变、基因敲除等手段制备危险基因变异的斑马鱼突变体，观察基因变异对血管功能的影响。本课题从分子细胞水平、模式生物研究到群体研究，互相印证，将有助于阐明血管发育缺陷在脑中风发病中的分子遗传机制，提供新靶标。

血管壁病变是脑中风发病的重要病理基础，是老龄化与环境因素的共同作用靶点，本项目采用分子遗传、模式生物和人群队列研究相结合的策略，从2个角度研究血管发育缺陷在脑中风发病中的分子遗传机制：（1）血管发育相关基因beta-Pix等基因对斑马鱼血管功能影响的研究，并利用beta-pix表达降低的fn40a突变体斑马鱼筛选抑制脑出血的小分子药物，结果发现beta-pix表达降低的fn40a突变体斑马鱼血管稳定性下降，通透性增加，MMP9表达上调，通过化学小分子出血抑制剂的筛选，找到抗真菌药物咪康唑具有明显的出血抑制效果，为脑出血的防治提供了新靶标。（2）利用人血管内皮细胞分析鉴定与染色体端粒异常和细胞老化相关的microRNAs，阐明microRNAs在动脉粥样硬化血管病变中的作用。通过传代培养，建立血管内皮细胞老化表型，采用Microarray 分析HUVECs老化过程中miroRNAs的差异表达，结果发现microRNA-216a 在老化内皮细胞的表达显著增加，细胞功能实验显示microRNA 216a可抑制内皮细胞的增殖、迁移等功能。本项目研究结果发表SCI论著4篇，参加国际学术会议交流3次，申请国内专利一项“咪康唑在预防血管破裂中的应用”，专利申请号为2016104070178。培养硕士研究生2名，博士研究生2名。