摘要
目的探讨NODAL信号通路在室间隔缺损形成中的潜在作用。方法应用荧光定量逆转录聚合酶链反应、蛋白免疫印迹方法分别检测室间隔缺损胎儿心脏组织中NODAL基因的表达(以同胎龄胎儿心脏组织为对照组)。分别构建SMAD2、CITED2基因表达质粒及NODAL基因荧光素酶报告基因质粒,并通过荧光素酶报告基因系统和免疫共沉淀实验对SMAD2、CITED2与NODAL基因之间相互作用进行研究。结果室间隔缺损胎儿心脏组织中NODAL基因表达较正常对照组降低,差异有显著性意义(P<0.01)。荧光素酶报告分析表明SMAD2和CITED2分别能上调NODAL基因表达,CITED2和SMAD2同时表达时NODAL基因的启动子活性呈4.3倍上调。免疫共沉淀实验表明CITED2与SMAD2存在于同一转录复合物中,共同调控NODAL基因的表达。结论初步确定NODAL信号通路异常与室间隔缺损发生具有相关性,为先天性心脏病的诊断、治疗、预防、预后及风险预测提供理论依据。
Objective To explore the potential role of the NODAL signaling pathway in the formation of the ventricular septal defect.Methods RT-qPCR and western blotting was used to examine the expression of NODAL mRNA in the normal and ventricular septal defect fetal heart tissues （with gestational age fetal heart tissue as control group）.SMAD2,CITED2 expression plasmid and NODAL gene luciferase reporter plasmid were constructed.The study on the interaction between SMAD2,CITED2 and NODAL were performed using Luciferase reporter gene system and immune co-precipitation experiments.Results NODAL gene expression in ventricular septal defect fetal heart tissue decreased compared with normal control group,and the difference was significant （P ＜ 0.01）.The luciferase reporter analysis showed that SMAD2 and CITED2 can up-regulate NODAL gene promoter activity.When the CITED2 and SMAD2 expression was at the same time,the promoter activity of NODAL genes was 4.3 times increased.The co-immunoprecipitation experiments showed that CITED2 and SMAD2 existed in the same transcriptional complex,which could co-regulate the NODAL gene expression.Conclusion It is initially identified that NODAL signaling pathway is related with the occurrence of abnormal ventricular septal defect,which will provide a theoretical basis for the diagnosis,treatment,prevention,prognosis and risk prediction of congenital heart disease.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2013年第9期781-784,788,共5页
Journal of China Medical University
基金
教育部博士学科点专项科研基金(20092104110011)
辽宁省科学技术计划(2011225017)
国家自然科学基金(81300511)
