GATA4基因对心内膜垫发育中相关蛋白表达作用的实验研究

Experimental studies on the role of GATA4 in the endocardial cushions development

目的 研究GATA4基因在心内膜垫发育过程中的作用.方法 构建GATA4野生型和突变型真核表达载体,利用脂质体Lipofectamine 2000介导重组质粒转染Hela细胞,分为Hela组、HelaGATA4组、HelaH436Y组、HelaNull组,应用实时PCR和Western blot检测转录因子GATA4、Sox9和Scleraxis及细胞外基质Aggrecan、Tenascin的mRNA和蛋白相对表达水平变化.结果 HelaGATA4组、HelaH436Y组、HelaNull组和Hela组GATA4基因mRNA相对表达水平分别为310.83±2.39、146.35±1.74、0.94±0.32和1.00±0.28 （F =72.508,P＜0.05）,且HelaGATA4组、HelaH436Y组均分别高于HelaNull组和Hela组（P均＜0.05）,HelaH436Y组低于HelaGATA4组（P＜0.05）;GATA4蛋白表达水平与mRNA变化一致.HelaGATA4组及Hela436Y组Sox9、Scleraxis、Aggrecan、Tenascin基因的mRNA和蛋白表达水平明显高于Hela组（P均＜0.05）,但它们在HelaH436Y组的表达水平低于HelaGATA4组（P均＜0.05）.结论 GATA4-H436Y突变使GATA4基因转录活性降低从而对下游基因的转录激活作用减弱,为阐明GATA4基因在心内膜垫发育成瓣膜中的作用提供了一定的理论基础.

Objective To investigate the role of GATA4 gene in the endocardial cushions development.Methods Target gene eukaryote expression vectors were constructed by pcDNA3.1 （-） vector plasmid,and were identified by DNA sequence analysis.Recombinant plasmids were transfected into Hela cells with lipofectamine 2000,meanwhile Hela cells transfected with empty vector or those without transfection served as transfection control group and blank control group,respectively.Real-time PCR and Western blot were performed to detect the relative expression of mRNA and protein of transcription factors GATA4,Sox9,Scleraxis and ECM proteins Aggrecan,Tenascin in each group.Results The relative mRNA expression of GATA4 in experimental group was significantly higher than in transfection control group and blank control group.GATA4 mRNA expression in HelaGATA4、Hela436Y 、HelaNull and Hela group was 310.83 ± 2.39,146.35 ± 1.74,0.94 ± 0.32,1.00 ± 0.28,respectively （F =72.508,P ＜ 0.05）.Western blot results were consistent with the results obtained by qRT-PCR.The relative mRNA and protein expressions of Sox9,Scleraxis,Aggrecan and Tenascin in both experimental groups were significantly higher than that in transfection control group and blank control group （P ＜ 0.05）,and above gene expressions were significantly downregulated in GATA4H436Y group,while they were similar between transfection control group and blank control group （all P ＞ 0.05）.Conclusions GATA4 H436Y mutation reduces it＇s transcriptional activation,which might serve as a theoretical framework to demonstrate the roles of GATA4 gene in endocardial cushion development.