沉默MagT1影响大鼠心肌细胞内Mg2+水平和细胞凋亡的研究

目的 使用siRNA沉默大鼠心肌细胞的镁离子转运蛋白1(MagT1),检测细胞内Mg2+浓度变化和细胞凋亡情况.方法 设计并合成MagT1 siRNA序列,转入原代培养大鼠心肌细胞沉默MagT1,使用反转录PCR和Western blot检测MagT1mRNA和蛋白表达情况,荧光显微镜检测细胞内Mg2+浓度变化情况,流式细胞法检测细胞凋亡情况.结果 相比阴性siRNA组,MagT1 siRNA转染大鼠心肌细胞48 h,MagT1 mRNA的沉默效率为51.83％ (P＜0.05),MagT1蛋白的沉默效率为56.75％(P＜0.05),胞内Mg2+浓度降低29.13％ (P＜0.05),细胞凋亡率为31.18％ (P＜0.01);转染60 h,MagT1 mRNA的沉默效率为86.91％ (P＜0.01),MagT1蛋白质的沉默效率为83.85％ (P＜0.01),细胞内Mg2+浓度降低41.32％ (P＜0.01),细胞凋亡率为40.61％ (P＜0.01).结论 MagT1被显著沉默后,细胞内Mg2+浓度显著降低,细胞凋亡显著提高,细胞生命活动受到较大影响.

Effect of silencing MagT1 on the content of magnesium ion and apoptosis in rat cardiomyocytes

Objective The changes of Mg2+ concentration and cell apoptosis were detected by using siRNA to silence MagT1 in rat cardio myocytes.Methods MagT1 siRNA sequences were designed and synthetized,then transfected into primary cultured rat myocardial cell for silencing MagT1.The expression of MagT1 mRNA and protein were detected by RT-PCR and Western blot.The changes of Mg2+ concentration in the cells were detected by fluorescence microscopy.Flow cytometry (FCM) was used to detect the cell apoptosis.Results Compared with negative siRNA group,MagT1 siRNA transfected rat cardiomyocytes after 48 h,MagT1 mRNA silence efficiency was 51.83 ％ (P＜0.05),the silence of MagT1 protein efficiency was 56.75 ％ (P＜0.05),intracellular Mg2+ concentration was reduced by 29.13％ (P＜0.05),the apoptosis rate was 31.18％ (P＜0.01);MagT1 siRNA transfected rat cardiomyocytes after 60 h,MagT1 mRNA silence efficiency was 86.91％ (P＜0.01),the silence of MagT1 protein efficiency was 83.85％ (P＜0.01),intracellular Mg2+ concentration was reduced by 41.32％ (P＜0.01),the apoptosis rate was 40.61％ (P＜0.01).Conclusion After the silencing of MagT1,the concentration of Mg2+ in the cells decreased significantly,the apoptotic rate increased significantly,cell life activities are greatly affected.