中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2015年
5期
437-441
,共5页
张艳丽%陈名武%陈冶%洪淑君
張豔麗%陳名武%陳冶%洪淑君
장염려%진명무%진야%홍숙군
心内膜垫缺损%GATA4基因%突变
心內膜墊缺損%GATA4基因%突變
심내막점결손%GATA4기인%돌변
Endocardial cushion defects%GATA4 transcription factor%Mutation
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.
目的 研究GATA4基因在心內膜墊髮育過程中的作用.方法 構建GATA4野生型和突變型真覈錶達載體,利用脂質體Lipofectamine 2000介導重組質粒轉染Hela細胞,分為Hela組、HelaGATA4組、HelaH436Y組、HelaNull組,應用實時PCR和Western blot檢測轉錄因子GATA4、Sox9和Scleraxis及細胞外基質Aggrecan、Tenascin的mRNA和蛋白相對錶達水平變化.結果 HelaGATA4組、HelaH436Y組、HelaNull組和Hela組GATA4基因mRNA相對錶達水平分彆為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組、HelaH436Y組均分彆高于HelaNull組和Hela組(P均＜0.05),HelaH436Y組低于HelaGATA4組(P＜0.05);GATA4蛋白錶達水平與mRNA變化一緻.HelaGATA4組及Hela436Y組Sox9、Scleraxis、Aggrecan、Tenascin基因的mRNA和蛋白錶達水平明顯高于Hela組(P均＜0.05),但它們在HelaH436Y組的錶達水平低于HelaGATA4組(P均＜0.05).結論 GATA4-H436Y突變使GATA4基因轉錄活性降低從而對下遊基因的轉錄激活作用減弱,為闡明GATA4基因在心內膜墊髮育成瓣膜中的作用提供瞭一定的理論基礎.
목적 연구GATA4기인재심내막점발육과정중적작용.방법 구건GATA4야생형화돌변형진핵표체재체,이용지질체Lipofectamine 2000개도중조질립전염Hela세포,분위Hela조、HelaGATA4조、HelaH436Y조、HelaNull조,응용실시PCR화Western blot검측전록인자GATA4、Sox9화Scleraxis급세포외기질Aggrecan、Tenascin적mRNA화단백상대표체수평변화.결과 HelaGATA4조、HelaH436Y조、HelaNull조화Hela조GATA4기인mRNA상대표체수평분별위310.83±2.39、146.35±1.74、0.94±0.32화1.00±0.28 (F =72.508,P＜0.05),차HelaGATA4조、HelaH436Y조균분별고우HelaNull조화Hela조(P균＜0.05),HelaH436Y조저우HelaGATA4조(P＜0.05);GATA4단백표체수평여mRNA변화일치.HelaGATA4조급Hela436Y조Sox9、Scleraxis、Aggrecan、Tenascin기인적mRNA화단백표체수평명현고우Hela조(P균＜0.05),단타문재HelaH436Y조적표체수평저우HelaGATA4조(P균＜0.05).결론 GATA4-H436Y돌변사GATA4기인전록활성강저종이대하유기인적전록격활작용감약,위천명GATA4기인재심내막점발육성판막중적작용제공료일정적이론기출.
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.
