摘要
目的探讨β-连环蛋白抑制基因2(DACT2)过表达对H9C2心肌细胞心力衰竭标志物的调控作用及其分子机制.方法以Ad-DACT2-绿色荧光蛋白(GFP)及其对照空载体Ad-CON177-GFP腺病毒液感染H9C2心肌细胞后,利用荧光显微镜观察H9C2心肌细胞的感染效率;利用实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质印迹法(Western blot)检测DACT2过表达效率;RT-qPCR检测DACT2过表达后心力衰竭标志物α-肌球蛋白重链(α-MHC)、β-肌球蛋白重链(β-MHC)、心房利钠肽(ANP)、脑利钠肽(BNP)和心钠素(ANF)的表达改变;Western blot检测DACT2过表达后Wnt/β-连环蛋白(β-catenin)和转化生长因子-β(TGF-β)/Smad通路关键蛋白表达变化.两组间比较采用独立样本t检验.结果绿色荧光显示细胞感染成功;与空载组比较,DACT2过表达组的mRNA和蛋白水平均明显增加(t=11.291,t=31.469,P<0.01),提示过表达模型构建成功;心力衰竭标志物oα-MHC(t=3.922,P< 0.05)、ANP(t=5.895,P<0.01)、BNP(t=30.195,P <0.01)和ANF(t=8.074,P<0.01)的表达水平明显降低,β-MHC(t=1.553,P>0.05)的表达水平无明显变化;DACT2过表达后Smad4的表达(=9.510,P<0.05)明显上调,其他因子的表达无显著改变.结论 DACT2通过上调Smad4抑制心力衰竭标志物的表达.
Objective To investigate the regulation of Dishevelled-binding antagonist of β-catenin 2 (DACT2) overexpression on H9C2 cardiomyocyte heart failure markers and its molecular mechanism by infecting H9C2 cardiomyocytes. Methods Ad-DACT2-green fluorescent protein (GFP) and its control empty vector Ad-CON177-GFP adenovirus were infected with H9C2 cardiomyocytes. The infection efficiency of H9C2 cardiomyocytes was observed by fluorescence microscope. The overexpression efficiency of DACT2 was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting;RT-qPCR for detecting DACT2 overexpression of heart failure markers α-myosin heavy chain (α-MHC),β-myosin heavy chain (β-MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and natriuretic peptide type A (ANF) expression changes;Western blotting was used to detect key protein expression changes of Wnt/β-Catenin and transforming growth factor-β(TGF-β)/Smad pathway after DACT2 overexpression. Independent sample t test was used for comparison between the two groups. ResultsThe green fluorescence showed successful cell infection. Compared with the empty group, the mRNA and protein levels of the DACT2 overexpression group were significantly increased (t=11.291, t=31.469, P<0.01), suggesting that the overexpression model was successfully constructed;Heart failure markers the mRNA expression levels of α-MHC (t=3.922, P<0.05), ANP (t=5.895, P<0.01), BNP (t=30.20, P<0.01) and ANF (t=8.074, P<0.01) was significantly decreased, the expression level of β-MHC was not significantly changed (t=1.553, P>0.05). The expression of Smad4 was significantly up-regulated after DACT2 overexpression (t=9.510, P<0.05), and the expression of other factors was not significantly changed. Conclusion DACT2 inhibits the expression of heart failure markers by up-regulating Smad4, suggesting that DACT2 may be a potential therapeutic target for heart failure diseases.
作者
黄加星
陈光献
黄穗青
冯康倪
李嘉文
侯健
吕林华
吴钟凯
Huang Jiaxing;Chen Guangxian;Huang Suiqing;Feng Kangni;Li Jiawen;Hou Jian;Lyu Linhua;Wu Zhongkai(Department of Cardiac Surgery,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China;NHC Key Laboratory of Assisted Circulation,Sun Yat-sen University,Guangzhou 510080,China NHC Key Laboratory of Assisted Circulation,Sun Yat-sen University,Guangzhou 510080,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第10期1819-1821,共3页
Chinese Journal of Experimental Surgery
基金
国家重点研发计划(2017YFC1105000)
国家自然科学基金(81770319).
