摘要
目的探索Sirt4在大鼠原代心肌细胞中对GATA结合蛋白4(GATA-binding protein 4,GATA4)活性的影响。方法构建过表达Sirt4(Ad-Sirt4)以及干扰Sirt4表达(Ad-sh Sirt4)的腺病毒并且在体外培养大鼠原代心肌细胞,将构建好的腺病毒及其相应的对照腺病毒(Ad-GFP/Ad-U6)分别感染大鼠原代心肌细胞,感染24 h后用PBS或1μmol/L血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)处理细胞48 h。采用Western blot及Real-time PCR检测GATA4的表达,通过免疫荧光及细胞核分离法检测GATA4的核转运,利用双荧光素酶报告基因法检测GATA4下游分子心房利钠肽(atrial natriuretic peptide,ANP)、脑利钠肽(brain natriuretic peptide,BNP)的启动子活性。结果 Sirt4在大鼠原代心肌细胞中不会影响GATA4的蛋白及mRNA水平(P>0.05)。与对照组比较,Sirt4过表达显著增加AngⅡ诱导的GATA4磷酸化,而干扰Sirt4表达降低GATA4的磷酸化水平。在大鼠原代心肌细胞中下调Sirt4表达可以明显抑制AngⅡ引起的GATA4入核;Sirt4过表达显著增加GATA4下游分子ANP、BNP的启动子活性(P<0.01)。结论 Sirt4可以在大鼠原代心肌细胞中促进AngⅡ对GATA4的激活效应。
Objective To determine the effect of Sirt4 on the activity of GATA-binding protein 4 (GATA4) in neonatal rat cardiomyocytes. Methods The AdEasy Vector kit was used to construct an adenoviral vector expressing Sift4 (Ad-Sirt4) and an adenoviral vector for knockdown of Sirt4 (AdshSirt4). Neonatal rat cardiomyocytes were cultured in DMEM supplemented with 10% FBS, and then infected with the recombinant adenoviruses respectively for 24 h. After that, cardiomyocytes were treated with PBS or 1 μmol/L angiotensin Ⅱ (Ang Ⅱ) for 48 h. The expression of GATA4 at mRNA and protein levels was detected by Real-time PCR and Western blotting respectively. Immunofluorescence assay and nucleus isolation kit were used to examine the nuclear transport of GATA4. The promoter activities of GATA4 downstream molecules, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), were detected by dual-luciferase reporter gene assay. Results Sirt4 had no effect on the protein and mRNA levels of GATA4 in neonatal rat cardiomyocytes (P 〉 0.05). Compared with the control, Sirt4 overexpression significantly increased Ang Ⅱ- induced GATA4 phosphorylation, whereas Sirt4 knockdown reduced the phosphorylation of GATA4. Sirt4 knockdown dramatically inhibited Ang Ⅱ-induced nuclear transport of GATA4. Sirt4 overexpression markedly increased the promoter activities of GATA4 downstream molecules (ANP and BNP, P 〈 0.01 ). Conclusion Sirt4 is able to promote Ang Ⅱ-induced GATA4 activation in neonatal rat cardiomyocytes.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2016年第14期1603-1608,共6页
Journal of Third Military Medical University