利用小鼠模型研究流感病毒导致心脏发育缺陷发生的分子机制

Clinical investigation data showed influenza inflection during the first trimester of pregnancy increase the risk of congenital heart disease (CHD), however, the related molecular mechanism remains unclear. Based on three influenza virus-adapted mouse models of H1N1, H5N1 and H9N2 strains, we found that the H1N1 strain has a direct infection on mice embryo heart tissue and caused a series of cardiac births defects such as AVSD. Recent studies hint NS1 protein binding on promoter of dvl2 gene may lead to mouse heart development anomalies through WNT/β-catenin signaling pathway. Firstly, this project plan to study the common and different CHD characteristics among influenza strains using microscopy dissection, ultrasound scanning and hematoxylin-eosin staining. After genome expression profiling scanning of heart tissue infected by influenza strains, we will investigate molecular mechanism of transcription between NS1 and dvl2 by ChIP and EMSA. Our aim is to find the underlying associations between WNT/β-catenin expression levels at different cardiac development stages and CHD in vivo. Our results will provide the directly experimental evidences，and clinic fundamentals of diagnosis, prevention and therapy on CHD caused by influenza inflection in pregnancy.

临床调查表明，孕早期流感病毒感染使先心病发生风险显著上升，但其分子机制尚不清楚。申请人在建立流感病毒H1N1、H5N1和H9N2鼠适应株模型基础上，发现H1N1流感病毒可直接感染小鼠胚胎心脏并导致多种先心病发生；国际和申请人研究进展提示，H1N1流感病毒NS1蛋白可能通过与dvl2基因启动子结合，调控WNT/β-catenin信号并导致小鼠心脏发育畸形。申请人拟进一步运用这些鼠适应株模型，采用显微解剖、超声影像、表达谱测序、ChIP、β-catenin报告基因体内示踪等方法，筛选鉴定流感病毒不同亚型致先心病发生的普遍性与特异性；研究NS1蛋白对dvl2基因的转录调控机制；H1N1流感病毒感染下，胚胎心脏发育不同阶段WNT/β-catenin信号表达变化与先心病表型的关联关系。研究结果将首次提供流感病毒致先心病发生的直接证据，为临床诊断、预防及治疗流感病毒导致的先心病提供工作基础。

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。本项目在建立流感病毒H1N1、H5N1和H9N2鼠适应株模型基础上，采用显微解剖、超声影像、组织切片的方法鉴定出了流感病毒感染导致先心病发生的房间隔缺损、单心房心室、室间隔缺损、右心发育不全、左心发育不良、三尖瓣闭锁、右心室双流出道、心肌臂薄、以及心肌组织结构疏松等多种表型。通过表达谱测序方法，筛选得到WNT信号途径和BMP信号途径相关及Bmp4、Gata4、Tbx5和Nkx2.5基因与流感病毒导致的CHD相关。采用ChIP、EMSA方法，研究了NS1蛋白对dvl2基因的转录调控机制。利用染色质免疫沉淀测序（ChIP-seq）和荧光实时定量聚合酶链反应技术（real-time PCR）筛选出了19个H1N1-NS1病毒蛋白结合区域临近的心脏功能基因。通过胶原凝胶外植体培养实验，观察到流感病毒抑制房室垫内皮-间质转化（EMT）过程中的细胞迁移，Real-time PCR和免疫荧光方法检测了参与EMT的信号成员和Bmp4、Gata4、Tbx5和Nkx2.5基因表达。进一步利用小鼠模型，检测了胚胎早期发育不同时期心脏组织中，WNT信号成员与心脏发育关键基因Bmp4、Gata4、Tbx5和Nkx2.5的表达，发现上述心脏发育标志基因和EMT标志物的表达变化与EMT进程受抑具有一致性。本项目结果说明流感病毒可能通过NS1蛋白对dvl2基因调控WNT信号影响EMT导致心脏结构发育缺陷的发生。研究结果首次提供了流感病毒致先心病发生的直接证据，为临床诊断、预防及治疗流感病毒导致的先心病奠定了基础。