用低浓度过氧化氢建立心肌细胞氧化损伤模型

Experimental model of H_2O_2-induced oxidative injury in cardiomyocytes

目的 观察低浓度 (10 0μm ol· L- 1 )过氧化氢对心肌细胞的时间损伤效应 ,探讨在细胞水平建立心肌氧化损伤模型 .方法 胰酶消化法分离培养心肌细胞 ,随机分为两组 :H2 O2 处理组和对照组 .在处理后不同的时间点 ,倒置显微镜下观察心肌细胞每分钟搏动频率 ;硫代巴比妥酸法检测心肌细胞脂质过氧化代谢产物丙二醛 (MDA) ;生化分析仪检测培养液中乳酸脱氢酶 (L DH) ;台盼蓝排斥试验检测细胞的存活率 .结果 H2 O2 处理后细胞搏动频率先有一个增加过程 ,而后迅速下降直至停止搏动 ;细胞 MDA的含量随作用时间的延长逐渐升高 ,30 min开始与对照组均差异显著 (P<0 .0 1) ;L DH漏出量也逐渐增加 ,40 min后与对照组均差异显著 (P<0 .0 1) ;H2 O2 处理 30 min后细胞存活率就开始下降 ,与对照组相比组间差异显著 (P<0 .0 1) .结论 10 0 μmol· L- 1的H2 O2 随着时间的延长表现出明显的细胞损伤效应 。

AIM To investigate the time course of H 2O 2 induced injury in cardiomyocytes and establish an experimental model of oxidative injury by using cultured neonatal rat ventricular myocytes. METHODS Ventricular myocytes were isolated from neonatal mice and cultured for 4~6 days. Cells were divided into 2 groups randomly: control group and H 2O 2 (100 μmol·L -1 ) group. After cells were exposed to H 2O 2,an Olmpus inverted phase contrast microscope was used to observe the contraction frequency of cells at different time points. The malondialdehyde (MDA) content in cardiomyocytes was determined by measuring thiobarbituric acid reactive substances to monitor lipid peroxidation. The activity of cytoplasmic enzyme lactate dehydrogenase (LDH) that was released into the culture media was assayed to indicate alternation in the integrity of the sarcolemmal membrane. Trypan blue exclusion was used to detect the cell viability. Cellular injury was evaluated in terms of the changes of contraction frequency, MDA content, LDH leakage and cell viability. RESULTS When cardiomyocytes were treated by H 2O 2, their contraction frequency increased significantly during the first 30 minutes, then decreased sharply and stopped in the end. Compared with the control group, MDA content, LDH leakage and cell death were significantly elevated when cells were treated respectively by H 2O 2 ( P <0.01). CONCLUSION The low concentration of H 2O 2 caused a significant time dependent injury in cells. This cultured primary neonatal mouse cardiomyocyte system provides a valuable tool to directly study cellular events of cardiac oxidative injury.