平面细胞极性途径调控对心肌细胞迁移、心肌组织构建及重塑影响的研究

Congenital heart disease (CHD) ranks the first in the birth defects. For this reason, applying epigenetic theory to study the mechanisms of clinical disease at the gene expression level might have great significance in scientific theory of primary prevention of congenital heart disease. Heart development involves the order of expression of numerous genes during embryonic development. Normal development of embryon depended on the finely tuned regulation of gene transcription and acetylation of histone plays an important role in this process. During the embryonic cardiac development, myocardial cell directional migration plays a vital role in atrioventricular septal closure and outflow tract formation, and histone acetylation modification also precisely regulates myocardial cell migration under the action of planar cell polarity (PCP) pathway. Our preliminary application chose mice to study the influence of histone acetylation imbalance in the PCP pathway to the expression and regulation of related genes and the expression and regulation, interfered with histone acetylation / deacetylation modified dynamic balance, successfully established animal model of congenital heart disease, but its mechanism still need further exploration. By means of epigenetics research tools, this project focus related genes ( HDACs, Vangl2 and Scrib ) expression regulation of histone acetylation modification imbalance in the PCP pathway, as well as myocardial cell migration movement and polar distribution of cytoskeletal proteins in newborn mouse, trying to reveal the important pathogenesis of CHD, and than makes some exploration to study the underlying cause of CHD with a new perspective, which may not only lay a solid experimental and theoretical basis for the prevention of CHD but also provide a new opinion on etiology research of the environmental factors lead to the etiology of congenital malformations.

先天性心脏病（先心病）居出生缺陷首位，其发病机制探索对先心病一级预防有重要意义。心脏胚胎发育过程中众多基因的顺序精确转录调控是胚胎正常发育的必要前提，而组蛋白乙酰化修饰是调控基因转录的重要途径。心脏胚胎发育中，心肌细胞定向迁移在房室间隔闭合与流出道形成中作用至关重要，组蛋白乙酰化修饰同样精确调控平面细胞极性（PCP）途径作用下的心肌细胞迁移。课题组前期应用药物干扰组蛋白乙酰化/去乙酰化修饰的动态平衡，成功建立了先心病动物模型，但其机制尚需深入探索。本项目利用表观遗传学的研究工具，对组蛋白乙酰化修饰失衡对PCP途径相关基因（HDACs、Vangl2及Scrib）表达调控的影响，以及新生小鼠心肌细胞迁移运动轨迹、骨架蛋白极性分布变化等进行研究，力图在调控基因表达层面，从心肌细胞迁移的全新角度揭示先心病的重要发病机制，为先心病的病因预防工作及环境致畸的病因学研究提供一条崭新的思路。

基因的顺序精确转录调控是心脏胚胎正常发育的必要前提，而组蛋白乙酰化修饰是调控基因转录的重要途径。心肌细胞定向迁移在胚胎心脏房室间隔闭合与流出道形成中作用至关重要，组蛋白乙酰化修饰同样精确调控平面细胞极性（PCP）途径作用下的心肌细胞迁移。本项目研究了组蛋白乙酰化修饰失衡致PCP途径相关基因（HDACs、Vangl2/Scrib/Rac1）变化的规律，结果显示，丙戊酸(VPA)作用新生小鼠心肌细胞后，去乙酰化作用下降，并呈现明显的量效关系；细胞极性消失，细胞内F-actin瓦解、边聚，细胞膜上板状与丝状伪足消失；HDAC1、HDAC3、Vangl2 mRNA于干预后24h、48h、72h均表达升高，HDAC2、Scrib mRNA于48h、72h表达升高（P＜0.05）；在蛋白水平，以上基因表达量虽随时间逐渐增加，但无统计学差异（P＞0.05）。VPA作用H9C2心肌细胞后，HDAC1、HDAC3、Vangl2、Scrib mRNA于24h、48h显著上调（P＜0.05）,而蛋白表达水平于48h、72h显著下调（P＜0.05），HDAC2、Rac1 mRNA水平无显著变化，但蛋白表达也显著下调（P＜0.05）。VPA作用孕鼠后，可诱导胎鼠先天心脏畸形，并且心脏畸形发生率与用药量和用药时间有相关性，其中以母鼠孕期第七天腹腔注射VPA 700 mg/kg 胎鼠心脏畸形发生率为最高，达 22.41% ，其中又以室间隔缺损常见。进一步对胚胎心脏的研究显示，HDAC1、HDAC3、Scrib于E11.5d基因表达下调（P＜0.05）；而HDAC1于E13.5d表达上调（P＜0.05），HDAC2、Vangl2、Rac1表达与对照组无统计学差异，而在蛋白表达水平上述各基因表达均无差异（P＞0.05）。综上结果提示，在心脏发育早期，VPA可以导致HDACs蛋白与基因的表达量发生变化，造成组蛋白乙酰化/去乙酰化失衡，而PCP途径与心脏发育相关的Vangl-2、Scrib基因与蛋白质的表达亦出现变化，从而导致胎鼠心肌细胞极性迁移阻滞和心肌组织重塑障碍，先天性心脏畸形发生，但具体的规律尚需深入研究。以上研究为先天性心脏病发病机制的研究提供了思路，对于先心病一级预防有着重要意义。