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目的研究心脏发育相关基因心肌转录因子NKX2.5和锌指转录因子GATA4在先天性心脏病(CHD)的相互作用关系。方法构建基因NKX2.5和GATA4真核表达质粒及脑钠肽启动子荧光素酶报告基因表达质粒,真核表达质粒经转染后,利用免疫共沉淀技术证明基因NKX2.5和GATA4两者是否有相互作用,通过荧光素酶分析技术分析基因NKX2.5和GATA4对脑钠肽启动子序是否存在协同激活作用。结果 p CMV-HA-NKX2.5与Pre-immune作用后蛋白表达量0,与Anti-Myc作用后蛋白表达量为3.63±1.23,与Input作用后蛋白表达量为2.95±1.05,NKX2.5可将GATA4沉淀下来;p CMV-Myc-GATA4与Pre-immune作用后蛋白表达量0,与Anti-Myc作用后蛋白表达量为5.63±2.06,与Input作用后蛋白表达量为5.75±2.86,GATA4可将NKX2.5沉淀下来;脑钠肽(BNP)启动子荧光表达质粒与两者共转染比分别单独转染NKX2.5和GATA4对脑钠肽启动子的激活作用更强。结论 NKX2.5和GATA4之间具有相互作用,在心脏发育过程中起着重要的协同作用。
Objective To study the relationship between cardiac transcription factor NKX2.5 and zinc finger transcription factor GATA4 in congenital heart disease (CHD). Methods The eukaryotic expression plasmids of NKX2.5 and GATA4 genes and luciferase reporter gene expression vector of brain natriuretic peptide promoter were constructed. After transfected with the eukaryotic expression plasmids, co-immunoprecipitation was used to confirm whether both genes NKX2.5 and GATA4 Interaction, by luciferase analysis of genes NKX2.5 and GATA4 on brain natriuretic peptide promoter whether synergistic activation exists. Results The protein expression level of p CMV-HA-NKX2.5 with Pre-immune was 0, the protein expression with Anti-Myc was 3.63 ± 1.23, and the protein expression with Input was 2.95 ± 1.05. NKX2.5 GATA4 was precipitated. The protein expression level of pCMV-Myc-GATA4 after pre-immune effect was 0, the protein expression level was 5.63 ± 2.06 after treated with Anti-Myc, and 5.75 ± 2.86 with Input. NKX2.5 was precipitated. The expression of the brain natriuretic peptide (BNP) promoter fluorescent plasmid and the co-transfection of NKX2.5 and GATA4 alone had stronger effect on the activation of brain natriuretic peptide. Conclusion There is an interaction between NKX2.5 and GATA4, which plays an important synergistic role in cardiac development.