先天性室缺EVC基因突变iPS系的建立及分子遗传机制研究

Ventricular septal defect (VSD) is the highest incidence of congenital heart disease(CHD) in recent years, domestic and international study found that genetic factors and CHD are closely related. Our previous study which based on the treatment of CHD in Jiangxi Province also found that two new mutations in the EVC are related with VSD.However, the mechanism of EVC in the development of heart progenitor cells in eartly human embryos is not clear.Restricted by ethic, researh in human congenital heart disease is difficult to achieve disease-specific and species-specific. In order to study the mechanism of the EVC in the pathogenesis of human VSD, we intends to establish disease-specific induced pluripotent stem cells (iPS) from skin cells with the mutant gene in patients, induce iPS into myocardial cells. With knockdown EVC and restore the wild-type EVC gene expression in iPS and myocardial cells differentiated from iPS by siRNA interference, detection of biological activity and the downstream protein expression may reveal relationship between EVC and transcription factor FGF2 and BMP-2, 4 and how EVC play a role in signalpathway in early embryonic regulation of cardiovascular specialization development. Exploring new intervention measures in pathogenesis of human VSD.

室缺是发病率最高的先天性心脏病,近年来国内外研究发现遗传因素与先心病发生密切相关。我们前期研究发现单纯室缺患者已知致病基因EVC的两个新的突变,但EVC在人类胚胎发育早期原始生心祖细胞发育中的作用机制目前尚不明确。受伦理等因素制约,以往对人类先天心脏病尤其是揭示先心病基因型和临床表型的相关性的研究很难做到疾病及种属特异性。为了研究EVC在人类室缺发病中的作用机制,本课题拟收集携带突变基因患者的皮肤细胞,建立疾病特异性诱导多能干细胞(iPS)系,并诱导为心肌细胞;通过对EVC基因进行siRNA干扰,再恢复野生型EVC基因的表达,观察iPS细胞及定向分化的心肌细胞的生物学活性及下游蛋白表达,研究EVC影响参与生心祖细胞特化发育的相关转录因子BMP-2,BMP-4,FGF2的表达情况,探明EVC在胚胎早期调节心血管特化发育的信号通路中的作用,为将来探索室缺新的干预措施提供有价值的理论依据。

以往对人类先心病或遗传性心脏病的研究工作多数利用动物模型（如转基因动物）或现有其他组织细胞株，但缺乏人类先心病疾病特异性，iPS为这类疾病研究的模型提供了可行的平台。本课题选择室缺患者为研究对象，收集获得EVC基因突变室缺患者尿液细胞，培养并诱导（再编程）为iPS细胞；利用iPS细胞多向分化潜能的特性，将其定向分化为有功能的心肌细胞，对室缺的发病机制进行探索。在本课题研究中，我们优化了iPS培养及心肌分化方法，建立了携带突变基因的iPS细胞系；并诱导分化成心肌细胞；通过探索EVC基因在影响原始心管发育信号通路中的作用，研究并发现EVC突变下调Hh通路活性的作用。利用iPS对先心病致病基因及易感基因进行发病机理的探索。解决了人类心脏细胞及标本来源的局限，避开了ESC建系的伦理问题，可实现先心病疾病发生的个体特异性，可为将来实现先心病治疗个体化研究寻找途径。