沉默MagT1诱发大鼠心肌细胞凋亡的机制及高Mg^(2+)对凋亡拯救的探究

Exploration of the Mechanism of Silencing MagT1-induced Cardiomyocyte Apoptosis in Rats and the Rescue of High Mg on Apoptosis

使用siRNA沉默大鼠心肌细胞的MagT1,检测细胞凋亡情况及机制、高浓度Mg^(2+)对于细胞凋亡的拯救情况。将MagT1 siRNA转入原代培养大鼠心肌细胞沉默MagT1,随后使用RT-PCR和Western blotting法检测MagT1 mRNA和蛋白质表达情况,荧光显微镜检测细胞内线粒体膜电位变化情况,流式细胞仪检测细胞凋亡和Caspase-9情况。相比阴性siRNA组MagT1 siRNA转染大鼠心肌细胞48 h后,MagT1 mRNA的沉默效率为52.06%(p<0.05),MagT1蛋白质的沉默效率为57.15%(p<0.05),细胞凋亡率为31.26%(p<0.01),高Mg^(2+)拯救组凋亡率为22.78%(p<0.01),线粒体膜电位水平降低22.03%(p<0.05) caspase-9表达水平升高7.93%(p>0.05);MagT1 siRNA转染大鼠心肌细胞60 h后,MagT1 mRNA的沉默效率为87.24%(p<0.01),MagT1蛋白质的沉默效率为84.45%(p<0.01),细胞凋亡率为40.64%(p<0.01),高Mg^(2+)拯救组凋亡率为26.62%(p<0.01)线粒体膜电位水平降低34.24%(p<0.01),caspase-9表达水平升高19.15%(p<0.05)。本研究发现MagT1被显著沉默后大鼠心肌细胞内Mg^(2+)浓度下降通过线粒体途径诱发了细胞凋亡,提升大鼠心肌细胞内Mg^(2+)浓度可以一定程度拯救凋亡。

Silencing MagT1 of rat cardiomyocytes with siRNA,then detecting the apoptotic status and mechanism,and the rescue effect of high concentration of Mg^(2+) on apoptotic cells.MagT1 siRNA was transfected into primary cultured rat cardiomyocytes to silence MagT1,and then the expression of MagT1 mRNA and protein was detected by RT-PCR and Western blotting,the change of mitochondrial membrane potential was detected by fluorescence microscopy.Apoptosis and Caspase-9 were detected by flow cytometry.Compared with negative siRNA group,48 hours after MagT1 siRNA transfection,the silencing efficiency of MagT1 mRNA was 52.06%(P<0.05),the silencing efficiency of MagT1 protein was 57.15%(p<0.05),the apoptosis rate was 31.26%(p<0.01),the apoptotic rate of high Mg^(2+) rescue group was 22.78%(p<0.05),the mitochondrial membrane potential level decreased by 22.03%(p <0.05),and the caspase-9 expression level increased by 7.93%(p>0.05).After 60 hours of transfection of Myocardial cells with MagT1 siRNA,the silencing efficiency of MagT1 mRNA was 87.24%(p <0.01),the silencingefficiency of MagT1 protein was 84.45%(p <0.01),and the apoptosis rate was 40.64%(p <0.01),the apoptotic rate of high Mg^(2+) rescue group was 26.62%(p<0.01),the mitochondrial membrane potential level decreased by 34.24%(p<0.01),and the expression of caspase-9 increased by 19.15%(p <0.05).In this study,we found that after MagT1 was significantly silenced,the decrease of Mg^(2+) concentration in rat cardiomyocytes induced apoptosis through mitochondrial pathway,and the increase of Mg^(2+) concentration in rat cardiomyocytes could rescue apoptosis to a certain extent.