液体石蜡封闭法建立大鼠心肌细胞缺氧/复氧损伤模型

目的:探讨液体石蜡封闭法建立大鼠心肌细胞H9C2缺氧/复氧损伤模型的可行性与稳定性。方法:将体外培养在六孔板中的大鼠心肌细胞H9C2随机分为对照组和实验组,对照组常规培养不做处理,实验组每孔细胞用高温灭菌的液体石蜡2mL封闭,造成缺氧环境,缺氧时间段分别为2、4、8、12h,处理结束后吸除石蜡,加入新鲜DMEM培养基,置二氧化碳培养箱中继续培养2h。超氧阴离子探针Dihydroethidium检测活性氧自由基含量变化;AnexinV/PI染色行流式细胞术检测不同时间缺氧/复氧处理后细胞凋亡率。结果:对照组细胞产生少量氧自由基;实验组随缺氧/复氧时间延长,氧自由基含量逐渐增加,缺氧2、4、8、12h细胞凋亡率分别为(0.76±0.15)%,(5.22±0.74)%,(10.36±2.07)%,(31.00±4.32)%,与对照组相比除缺氧2h组外其余三组均有显著性差异(P<0.05)。结论:液体石蜡封闭法建立H9C2缺氧/复氧损伤模型简单易行、重复性好。

Establishment of Myocardial Hypoxia/reoxygenation Injury Model in Rat with Liquid Paraffin Covering Method

Objective To investigate the feasibility and stability of establishing myocardial hypoxia/reoxygenation model with liquid paraffin covering method.Methods The H9C2 cells cultured in vitro were randomly divided into control group and experimental group.Control group was routinely cultured.The cells in experimental group were covered with sterilizing liquid paraffin for 2,4,8,12 h,respectively.Then replaced paraffin with DMEM medium and cultured for another 2 h in CO2 incubators.The reactive oxygen species content was observed by superoxide anion radical probe-Dihydroethidium.Flow cytometry was used to test the apoptotic rate at different time points.Results There were only few reactive oxygen species in control group and the content was increased with the prolonging of hypoxia/reoxygenation time;the apoptotic rate in experimental group were （0.76±0.15）%,（5.22±0.74）% ,（10.36±2.07）%,（31.0±4.32）% ,respectively.They were all significantly higer than that of control group（P0.05） excluding hypoxia for 2 h group.Conclusion The establishment of H9C2 hypoxia/reoxygenation model with liquid paraffin covering method is simple and practicable.