流感病毒H1N1通过BMP4-GATA4信号调控EMT发生致先心病发生的机制研究

Clinical epidemiology survey indicates that maternal influenza viral infection poses an increased risk of cardiac malformation in the fetuses, the underlying molecular mechanism remains unclear. Previous studies demonstrated that exposure of the early chick embryo to imidacloprid caused down-regulation of BMP4, GATA4 and NKX2.5 and improper process of EMT, thereby leading to malformation of heart tube. Congenital heart defects mouse model has been established by influenza virus infection on E7.5 pregnant mouse in our laboratory.By combination of such model and RNA-seq screening, we obtained a number of genes with significantly repressed expression, including BMP4, GATA4 and NKX2.5. Our preliminary work confirmed the sequencing results by qPCR. We therefore hypothesize that influenza virus H1N1 induced BMP4-GATA4 signal regulating EMT process may contribute to the development of congenital heart disease. The current project plans to apply mouse and cellular models and pathological specimen of human heart to explore the association between influenza virus H1N1 infection, EMT occurrence and cardiac birth defects, and to reveal the mechanism of H1N1 induced congenital heart disease via BMP-GATA4 signal regulating EMT process.

流行病学临床调查显示孕妇宫内感染流感病毒可以增加胎儿心脏发育畸形的风险，但其分子机制仍不清楚。国际上利用动物模型证实环境刺激可以抑制BMP信号成员BMP4、GATA4、NKX2.5等基因表达，从而干扰EMT发生导致心管发育畸形。申请人等利用流感病毒H1N1宫内感染导致胚胎心脏结构发育缺陷的小鼠模型，通过RNA-seq筛选流感病毒H1N1感染怀孕母鼠胚胎心脏组织的差异表达基因，得到表达显著下调的BMP4、GATA4、NKX2.5等基因；并进一步在小鼠模型中通过qPCR验证了测序筛选的结果。申请人据此假设，流感病毒H1N1通过BMP4-GATA4信号调控EMT进程致心脏结构发育缺陷的发生。本项目拟利用小鼠和细胞模型以及人体病理标本，研究流感病毒H1N1感染、EMT机制和心脏发育出生缺陷三者之间的关系，并证明H1N1通过BMP4-GATA4信号调控EMT进程致使心脏结构发育缺陷发生的分子机制。

临床流行病学调查显示孕妇宫内感染流感病毒可以增加胎儿心脏发育畸形的风险，但其分子机制仍不清楚。本课题组通过对感染流感病毒的孕母鼠的胚胎早期发育不同时期胚胎和胚胎心脏进行形态学观察，以探究流感病毒宫内感染和先天性心脏缺陷发生之间的关系；通过分子生物学手段，阐明BMP4-GATA4信号在宫内感染流感病毒后心脏结构发育畸形中的作用机制。其具体结果如下：（1）建立流感病毒感染怀孕的大鼠模型，证明流感病毒宫内感染和先天性心脏缺陷发生相关；（2）利用染色质免疫沉淀测序（ChIP-seq）、荧光实时定量聚合酶链反应技术（real-time PCR）等证明流感病毒可能通过BMP信号调控其下游基因表达造成心脏发育异常；（3）通过胶原凝胶外植体培养实验证明流感病毒感染可能介导BMP信号影响房室垫内皮-间质转化（EMT）的发生，从而导致心脏结构发育缺陷的发生。本研究为预防和治疗先心病提供理论和实验依据。