2%氧浓度下H_(9)C_(2)细胞缺氧/复氧模型的优化

Optimization of hypoxia/reoxygenation model of H_(9)C_(2) cells under 2%oxygen concentration

目的优化2%氧浓度下的H_(9)C_(2)细胞缺氧/复氧模型。方肉基于以往方法,将处于对数生长期的H_(9)C_(2)心肌细胞置于2%氧浓度的移动式三气培养箱中4、6、8、10、12 h,再分别复氧4h。用CCK-8法测定心肌细胞存活率,用比色法测定乳酸脱氢酶(LDH)活性。采用正交试验法优化细胞培养液条件(空白血清比例,含糖量),确定模型的最佳缺氧/复氧时间和培养条件。结果H_(9)C_(2)心肌细胞在缺氧4h/复氧4h、缺氧6h/复氧4h、缺氧8h/复氧4h、缺氧10h/复氧4h和缺氧12h/复氧4h时,心肌细胞存活率呈下降趋势;细胞上清液中LDH活性呈升高趋势。正交试验结果:20%血清含量;无糖培养基缺氧8 h、正常培养基复氧4 h为缺氧/复氧模型最优条件。结论优化的2%氧浓度下的H_(9)C_(2)细胞缺氧/复氧模型更趋稳定性、可控性、重复性。

Objective To optimize the hypoxia/reoxygenation model of H_(9)C_(2) cells under 2% oxygen concentration.Methods Based on the previous method,H_(9)C_(2) cardiomyocytes in logarithmic growth phase were incubated in a mobile three-gas incubator with 2% oxygen concentration for 4,6,8,10 and 12 h,and then reoxygenated for 4 h.The viability of cardiomyocytes was measured by CCK-8 method and the activity of lactate dehydrogenase(LDH)was measured by colorimetry.The orthogonal test method was used to optimize the cell culture fluid conditions(blank serum ratio,sugar content)to determine the optimal hypoxia/reoxygenation time and culture conditions for the model.Results The viability of H_(9)C_(2) cardiomyocytes decreased when hypoxia for 4h/reoxygenation for 4h,hypoxia for 6h/reoxygenation for 4h,hypoxia for 8h/reoxygenation for 4h,hypoxia for 1 Oh/reoxygenation for 4h and hypoxia for 12h/reoxygenation for 4h.The activity of lactate dehydrogenase in cell supernatant shows an increasing trend.Results of orthogonal test showed that 20% serum content,hypoxia in sugar-free medium for 8 hours and reoxygenation in normal medium for 4 hours were the optimal conditions for establishment of hypoxia/reoxygenation model.Conclusion The hypoxia/reoxygenation model of H_(9)C_(2) cells under the optimized 2% oxygen concentration is more stable,controllable,and reproducible.