内源性多肽YSGPTCH及其介导的Notch信号通路致室间隔缺损的作用机制研究

Peptide YSGPTCH is an endogenous polypeptide that has been found to be distinct from the normal control of fetal amniotic fluid in the ventricular septal defect by means of peptidomics. Previous studies showed that YSGPTCH was conserved between different species and was significantly higher in peripheral blood of children’s mother as well as in the amniotic fluid with ventricular septal defect. It reported that NOTC4, precursor protein of YSGPTCH, is involved in the cardiac development-related Notch signaling pathway. Our previous study found that peptide YSGPTCH significantly inhibited the differentiation of P19 cells into cardiomyocyte-like cells and resulted in abnormal cardiac development in zebra fish. The addition of YSGPTCH to P19 cells induced inhibition of Hey2 expression(downstream of Notch signaling pathway), suggesting that YSGPTCH may affect fetal cardiac development by regulating Notch-Hey signaling pathway. In this study, we intend to investigate in depth the role of YSGPTCH in myocardial differentiation and cardiac development. With the Notch-Hey signaling pathway as a clue, the mechanism by which YSGPTCH participates in the regulation of cardiac differentiation and cardiac development is to be demonstrated. Our aim is to elucidate the role and mechanism of this polypeptide with unknown function in embryonic heart development, and to provide a new diagnostic marker for early pregnancy screening for congenital heart disease.

多肽YSGPTCH是我们利用多肽组学发现的、在不同物种间高度保守、高表达于人室间隔缺损胎儿及其母亲外周血中、功能未知的一条内源性多肽。既往报道YSGPTCH的前体蛋白NOTC4参与了心脏发育相关的Notch信号通路。我们前期的研究发现，多肽YSGPTCH干预显著抑制P19细胞向心肌样细胞的分化，并可导致斑马鱼心脏发育畸形，多肽YSGPTCH加入P19细胞诱导分化后Notch信号通路信号下游Hey2表达受抑制，提示多肽YSGPTCH可能通过调控Notch-Hey信号通路而影响胎儿心脏发育。本研究拟深入探讨多肽YSGPTCH在心肌分化和心脏发育中的作用；并以Notch-Hey信号通路为线索，论证多肽YSGPTCH参与调控心肌分化和心脏发育的具体机制。阐明这一功能未知的多肽在胚胎心脏发育中的作用及机制，也可为先心病在妊娠早期筛查提供新的诊断标志物。

多肽YSGPTCH是我们利用多肽组学发现的、在不同物种间高度保守、高表达于人室间隔缺损胎儿及其母亲外周血中、功能未知的一条内源性多肽。既往报道YSGPTCH的前体蛋白NOTC4参与了心脏发育相关的Notch信号通路。通过本研究发现（1）在细胞水平：多肽YSGPTCH干预后，抑制P19细胞增殖，抑制P19细胞向心肌样细胞的分化，Notch信号通路信号下游Notch4、Hey2基因mRNA表达受抑制，提示多肽YSGPTCH可能通过调控Notch-Hey信号通路而影响心肌细胞发育。（2）在动物水平：我们将不同浓度的多肽注射到斑马鱼受精卵，斑马鱼能够产生明显的心脏畸形表型，随着多肽浓度的逐渐升高，心脏畸形的比例升高明显增高、心率随着浓度的升高逐渐上升、随着时间推移斑马鱼能够产生心率明显加快。与对照组相比，多肽干预组斑马鱼心房、心室扩大，心室壁变薄，心包水肿明显，随着时间的延长，斑马鱼存活率逐渐下降。HE染色及cmlc-EGFP标记荧光染色提示斑马鱼正常心房心室结构消失、异常转位、瓣膜发育不良。利用原位杂交技术我们检测到心脏中cmhc、vmhc的表达改变，表明不同浓度的YSGPTCH均可影响斑马鱼的胚胎发育，且呈剂量依赖性。以上均提示多肽YSGPTCH在心脏发育过程中存在着微调节作用，也可为先心病在妊娠早期筛查提供潜在的诊断标志物。