中心体蛋白290与小鼠圆锥动脉干畸形发病机制的相关性研究

Conotruncal defect is one of the most common and severest congenital cardiac anomalies, it accounts for 30% of all congenital heart disease. Although it has been demonstrated that cilium and cilia related signaling have an association with the pathogenesis of congenital heart disease, its exact mechanism still remains uncertain. Centrosomal protein 290 (CEP290) has been found to play an important role in the formation, construction and function of cilia. We previously have discovered a novel homozygous point mutation of CEP290 (c.T4670A，p.L1557X) as one of causative gene mutations in mouse conotruncal defects. In this study, we aim to investigate how mutations in CEP290 may cause conotruncal defects by analyzing deployment, proliferation, apoptosis and differentiation of second heart field and cardiac neural crest cells. In addition, studies will be conducted to determine if formation, length, construction of primary cilia are disrupted by cep290 gene mutation, and to explore if the expression level of CEP290 mRNA and protein, and localization of CEP290 in cilia are altered in the CEP290 mutant mouse embryonic fibroblasts. Furthermore, we plan to determine the role of CEP90 in Shh signaling. The main goal of this study is to elucidate the role of CEP290 in outflow tract development and how disruption of cep90 function contributes to conotruncal defect.

圆锥动脉干畸形是先天性心脏病最常见也是最严重的类型之一, 约占先天性心脏病的30%。已有研究表明：纤毛和由纤毛转导的细胞信号与先天性心脏病发生密切相关，但其具体机制并不清楚。中心体蛋白290 (CEP290)可能在纤毛形成、正常结构与功能的维持中发挥着重要作用。我们前期在对一个圆锥动脉干畸形小鼠家系进行研究时证实CEP290（c.T4670A，p.L1557X）基因纯合子突变可导致圆锥动脉干畸形的发生。本研究拟首先研究CEP290基因突变对小鼠胚胎发育流出道中第二生心区及心脏神经嵴细胞的分布，以及增殖、凋亡和分化的影响；其次，拟研究CEP290基因突变对初级纤毛形成率、纤毛长度以及结构的影响，以及对mRNA表达丰度以及蛋白表达水平、纤毛内定位产生的影响。第三，拟研究通过观察CEP290基因突变对Shh信号通路的影响，探讨CEP290通过影响Shh信号通路参与圆锥动脉干畸形发生的可能机制。

圆锥动脉干畸形是最常见、最严重的先天性心脏病之一，占所有先天性心脏病的30%。虽然已经证实纤毛及其相关信号与先天性心脏病的发病有关，但其确切机制仍不清楚。中心体蛋白290（CEP290）在纤毛的形成、结构和功能中起着重要作用。在本研究中，我们研究了由CEP290突变引起的圆锥动脉干畸形的疾病谱分布。我们分析了不同基因型小鼠胚胎发育流出道中第二生心区和心脏神经嵴细胞的分布、凋亡差异。发现初级纤毛的形成、长度、结构不受cep290基因突变的干扰。用RT-PCR和Western blot技术研究了突变体和野生小鼠Shh信号基因及蛋白的表达差异。结果表明，与野生型相比，CEP290突变胚胎小鼠胚胎及小鼠胚胎成纤维细胞Shh信号基因及蛋白的表达有显著差异。这项研究揭示了CEP290在心脏圆锥动脉干发育中的作用，以及cep90参与圆锥动脉干畸形的具体机制。