缺血再灌注对大鼠心肌H9c2细胞活性、凋亡的影响及其机制

Influence of ischemia reperfusion on viability and apoptosis of H9c2 myocardial cells and its mechanism

目的观察缺血再灌注对大鼠H9c2心肌细胞活性、凋亡的影响,并探讨其作用机制。方法 H9c2细胞分为A、B组,1×10~4/孔接种于96孔板,每组6个复孔。细胞贴壁生长24 h时B组吸弃原培养基,加入人工缺氧液100μL,置于含94%N_2、1%O_2、5%CO_2的三气培养箱中培养90 min;吸弃缺氧液,加入DMEM高糖培养基,置于正常培养箱中培养90 min备用。A组仅加入DMEM高糖培养基,置于正常培养箱中培养90 min备用。采用MTT法检测细胞活性,TUNEL法检测凋亡细胞,采用激光共聚焦显微镜观察并计算两组细胞活性氧簇（ROS）含量及线粒体膜电位（MMP）,采用Western blotting法检测心肌细胞热休克蛋白60（HSP60）、过氧化物氧化还原酶（Prx）、硫氧还原蛋白（Trx）、线粒体分裂蛋白（Fis1）。结果 A、B组心肌细胞活性分别为0.77±0.01、0.40±0.02,二者比较,P〈0.05。A、B组心肌细胞凋亡率分别为2.24%±0.12%、41.78%±1.43%,二者比较,P〈0.05。A组细胞ROS含量及MMP分别为0.58%±0.02%、1.12%±0.05%,B组分别为1.13%±0.05%、0.76%±0.01%,组间比较,P均〈0.05。与B组相比,A组心肌细胞HSP60、Fis1蛋白表达升高,Prx2、Trx1蛋白表达降低,P均〈0.05。结论缺血再灌注后H9c2细胞出现细胞活性下降、细胞凋亡,其机制可能与缺血再灌注促进细胞HSP60、Fis1蛋白表达,抑制Prx2、Trx1蛋白表达有关。

Objective To investigate the influence of ischemia reperfusion（ IR） on cell viability and apoptosis of rat myocardial cells H9c2 as well as the potential molecular mechanism. Methods H9c2 cells were divided into groups A（ control） and B（ IR） and were seeded in 96-well plates by 1 × 10~4/well. After adhering for 24 h,the culture medium of group B was replaced by 100 μL hypoxia buffer and the cells were incubated in 94% N_2,1% O_2,and 5% CO_2 for 90min. Then we discarded the hypoxia buffer,and incubated them in normal environment with 100 μL/well DMEM of high glucose medium for another 90 min. The cell viability was detected by MTT,apoptosis by TUNEL,mitochondrial membrane potential（ MMP） and reactive oxygen species（ ROS） by laser scanning confocal microscope. Furthermore,heat shock protein 60（ HSP60）,peroxiredoxin（ Prx）,thioredoxin（ Trx） and mitochondria fission protein（ Fis1） were detected by Western blotting. Results The cell viability of groups A and B was 0. 77 ± 0. 01 and 0. 40 ± 0. 02,respectively（ P〈0. 05）. The apoptosis rates in groups A and B were 2. 24% ± 0. 12% and 41. 78% ± 1. 43%（ P〈0. 05）. Intracellular ROS and mitochondrial membrane potential of group A was 0. 58% ± 0. 02% and 1. 12% ± 0. 05%,and was 1. 13% ±0. 05% and 0. 76% ± 0. 01% in the group B（ all P〈0. 05）. Compared with group A,the expression levels of HSP60 and Fis1 were increased,and the levels of Prx2 and Trx1 were decreased（ all P〈0. 05）. Conclusion After ischemia reperfusion,the cell viability decreases and the apoptosis increases in H9c2 cells,and its mechanism may be that ischemia reperfusion promotes the expression of HSP60 and Fis1 protein and inhibits the expression of Prx2 and Trx1 protein.