钠泵α2亚基对心肌缺血再灌注损伤的影响及其机制

发布时间：2018-02-10 04:38

本文关键词： 心肌缺血再灌注损伤钠泵内质网应激细胞凋亡　出处：《皖南医学院》2017年硕士论文　论文类型：学位论文

【摘要】：目的:在细胞水平上,探讨钠泵α2亚基对心肌缺血再灌注(ischemia reperfusion,IR)损伤的影响并进一步探讨钠泵信号通路是否通过内质网应激反应参与心肌缺血再灌注损伤。方法:原代心肌细胞培养成功后,在细胞水平上构建缺氧复氧模型。缺氧复氧过程中,对各组采用不同的干预处理。实验分四组:(1)钠钾泵α2亚基siRNA+缺氧复氧组(Na+-K+pumpα2siRNA+I/R)(2)阴性病毒+缺氧复氧组(control+I/R),(3)正常对照组(4)缺氧复氧组(I/R)。缺氧复氧完成后通过荧光显微镜观察细胞生长情况,荧光定量PCR检测各组钠钾泵α2亚基m RNA的表达;流式细胞仪,采用Annexin V-FITC/PI双染法检测细胞凋亡;采用Western blot检测内质网应激指标及信号分子GRP-78以及促凋亡蛋白CHOP表达水平。结果:1.荧光定量PCR检测钠钾泵α2亚基mRNA:与对照组相比,I/R组与control-I/R组细胞α2亚基mRNA含量降低(P0.05);与I/R组相比,钠钾泵α2si RNA-I/R组细胞m RNA明显含量降低(P0.05)2.流式细胞仪检测细胞凋亡率:与对照组相比,I/R组与control-I/R组细胞的凋亡率明显增高(P0.05),与I/R组相比,钠钾泵α2siRNA-I/R组细胞凋亡率亦明显增高(P0.05)3.western-bolt结果:与对照组相比,control-I/R组细胞内CHOP及GRP-78的蛋白表达量明显增高(P0.05);与I/R组相比,钠钾泵α2siRNA-I/R组细胞CHOP及GRP-78的蛋白表达量亦明显增高(P0.05)。结论:1、抑制钠泵α2亚基的活性会加重缺血再灌注细胞内质网应激、加重心肌细胞凋亡。2、钠泵α2亚基可能通过诱导内质网应激参与心肌细胞凋亡并参与心肌缺血再灌注损伤。
[Abstract]:Objective: at the cellular level, To investigate the effect of sodium pump 伪 2 subunit on myocardial ischemia reperfusion IRs and whether the sodium pump signaling pathway is involved in myocardial ischemia reperfusion injury through endoplasmic reticulum stress. Methods: primary myocardial cells were cultured successfully. Build a model of anoxia reoxygenation at the cellular level. Each group was treated with different intervention. The experiment was divided into four groups: 1) sodium and potassium pump 伪 2 subunit siRNA anoxic reoxygenation group (Na K pump 伪 2 siRNA I / R 2) negative virus hypoxia reoxygenation group control I / R (3) normal control group 4) hypoxia reoxygenation group (4) hypoxia reoxygenation group (I / R). Microscopic observation of cell growth, Fluorescence quantitative PCR was used to detect the expression of 伪 2 subunit m RNA, flow cytometry was used to detect apoptosis by Annexin V-FITC / Pi double staining. Western blot was used to detect endoplasmic reticulum stress, signal molecule GRP-78 and CHOP expression of apoptotic protein. Results: 1. Fluorescence quantitative PCR was used to detect 伪 2 subunit mRNAs of sodium and potassium pump: compared with control group, mRNA content of 伪 2 subunit in IPRR group and control-I/R group was decreased (P 0.05), compared with that in IR / R group. The content of m RNA in 伪 2si RNA-I/R group decreased significantly. Flow cytometry was used to detect the apoptosis rate. Compared with the control group, the apoptosis rate of the control-I/R group and the I / R group was significantly higher than that of the I / R group, and that of the I / R group was significantly higher than that of the I / R group, and that of the I / R group was significantly higher than that of the control group. The apoptosis rate of 伪 2siRNA-I / R group was also significantly higher than that of the control group. The results showed that the expression of CHOP and GRP-78 protein in the control group was significantly higher than that in the control group (P 0.05), and that in the I / R group was significantly higher than that in the I / R group. In sodium potassium pump 伪 2siRNA-I / R group, the protein expression of CHOP and GRP-78 was also significantly increased (P 0.05). Conclusion the inhibition of the activity of sodium pump 伪 2 subunit may aggravate the endoplasmic reticulum stress in ischemia-reperfusion cells. By inducing endoplasmic reticulum stress, sodium pump 伪 2 subunit may participate in myocardial apoptosis and myocardial ischemia-reperfusion injury.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54

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