P38信号通路在心梗后大鼠心肌钾通道重构的氧化还原调控机制

发布时间：2018-02-28 13:16

本文关键词： p38激酶硫氧还蛋白钾通道 Ito电流心肌梗死　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:p38激酶是细胞凋亡的关键调节因子,尤其在因为病理性应激所导致的心肌肥厚及坏死中,但是它对病理状态下心肌离子通道尤其是电压门控钾通道(Kv)的调控机制还并不清楚。所以本研究目的是进一步明确大鼠心梗(MI)6-8周后p38信号通路在电压门控钾通道(Kv)调控中的作用机理。方法:从河北医科大学实验动物中心购得雄性Sprague-Dawley(SD)大鼠。称得体重在180~200g的雄性Sprague-Dawley(SD)大鼠按照文献方法建立MI模型,在手术后6-8周,对大鼠进行麻醉,开胸分离出心脏,并使用Langendoff法通过冠状动脉进行胶原酶灌流,获得单一分离的心室肌细胞,在CO2培养箱中孵育4.5小时后,应用细胞膜片钳试验对样本电压门控钾通道(Kv)及Ito进行分析。本实验使用非放射性p38激酶检测试剂盒检测(细胞信号技术)p38激酶活性。结果:1使用免疫印迹法检测显示MI后心肌p38激酶活性明显增强,与对照组相比增加大约2-6倍(Control组:2.0±1.1,n=6;MI组:7.2±1.7,n=6;P0.05)。2把实验组分离提取的组织标本经过与p38激酶阻断剂SB20358015?M混合后4-5个小时,应用电生理技术检测Ito电流强度,可见其明显增高(SB203580+MI:25.6±1.0 p A/p F,n=10;MI组:15.9±2.1 p A/p F,n=10;P0.05)。3硫氧还蛋白(Trx)还原酶阻断剂金诺芬(AF)试剂明显阻断了SB203580对MI老鼠心肌Ito电流的明显的增强效果(MI+AF+SB203580:0.5±0.1,n=10),而AF对对照组Ito无明显影响,Trx能够显著增加Ito电流的强度,另外胰岛素样生长因子能够增加Trx的作用,能够增强Ito电流的强度。4免疫蛋白印记法显示经过p38阻滞剂SB203580溶解混合后的MI心肌细胞的Kv4.2通道蛋白表达量显著增加,即使并没有恢复到未处理组的水平,但是这与既往在MI心肌相关的研究中所了解到的Ito电流密度的改变是一致的。经过p38激酶阻断剂SB203580处理过的对照组的相关心肌的Kv4.2通道蛋白表达含量没有表现出明显增多的迹象,而基本上保持平衡。结论:缺血坏死导致心梗的心肌钾通道改变是能够调节的,能够通过激活p38信号通路促进钾通道的改变导致Ito改变。这一过程可被Trx、IGF-1调控。本研究结果表明,在MI心肌中,p38激酶活性明显增强,进一步降低Kv通道介导的Ito电流强度;抑制p38信号通路能够明显提高Kv通道蛋白表达,进而提高Ito电流强度,这一过程能被Trx、IGF-1系统调控。
[Abstract]:Objective: p38 kinases are key regulators of apoptosis, especially in hypertrophy and myocardial necrosis because of pathological stress caused by, but it is of cardiac ion channels under pathological conditions especially voltage-gated potassium channels (Kv) of the regulation mechanism is not clear. So the purpose of this study is to further clarify the rat myocardial infarction (MI) after 6-8 weeks of p38 signaling pathway in the voltage-gated potassium channel (Kv) in the regulation mechanism. Methods: male Sprague-Dawley purchased from the experimental animal center of Hebei Medical University (SD) in rats. Called weight in male Sprague-Dawley 180~200g (SD) rat MI model was established according to the methods of literature, in the 6-8 weeks after surgery, the rats were anesthetized, open chest isolated heart, and collagenase perfusion through coronary artery using the Langendoff method to obtain a single isolated ventricular myocytes, cultured in the CO2 box after 4.5 hours of incubation by cell membrane Clamp voltage gated potassium channel on the test sample (Kv) and Ito were analyzed. The experiments using non radioactive p38 kinase assay kit (cell signaling technology) p38 kinase activity. Results: 1 using immunoblotting assay showed that after MI myocardial p38 kinase activity increased significantly, compared with the control group increased about 2-6 times (group Control: 2 + 1.1, n=6; group MI: 7.2 + 1.7, n=6; P0.05).2 the experimental group extracted from tissue samples with p38 kinase inhibitor SB20358015? M 4-5 hours after mixing, the electrophysiological technique was used to detect Ito current intensity, can see the increased (SB203580+MI:25.6 + 1 p A/p F, n=10; group MI: 15.9 + 2.1 P A/p F, n=10; P0.05).3 thioredoxin reductase inhibitor (Trx) Jin Nuofen (AF) SB203580 of MI reagent significantly blocked the mouse myocardial Ito current obvious enhancement effect (MI+AF+ SB203580:0.5 + 0.1, n=10), while the AF of control group Ito no Obviously, Trx can Ito the current intensity increased significantly, and insulin-like growth factor can increase the effect of Trx, can enhance the strength of.4 Western blot method showed that after Ito current MI myocardial p38 blocker SB203580 dissolving the Kv4.2 channel protein expression was significantly increased, even if did not return to the untreated group the level of Ito, but the current density in the MI are aware of this and previous myocardial related research in the change is consistent. After blocking the Kv4.2 channel protein kinase p38 expression in myocardial control group treated by agent SB203580 showed no signs of a marked increase, and basically balance. Conclusion: ischemic necrosis leading to myocardial infarction is the change of potassium channel can be adjusted, can activate p38 signaling pathway to promote potassium channel changes lead to changes in Ito. This process can be Trx, IGF-1 Regulation and control. This study shows that in MI myocardium, p38 kinase activity is significantly enhanced, further reducing Kv channel mediated Ito current intensity. Inhibition of p38 signaling pathway can significantly improve Kv channel protein expression, and further enhance Ito current intensity, which can be regulated by Trx and IGF-1 system.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.22

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