葡萄糖/葡萄糖氧化酶诱导H9c2心肌细胞凋亡模型的建立

目的 研究不同浓度高糖（glucose,G）/葡萄糖氧化酶（glucose oxidase,GO）对H9c2细胞凋亡的影响,以确定建立H9c2细胞因糖自氧化所致凋亡模型的合适浓度.方法 在含33 mmol/L葡萄糖的高糖培养液中以不同浓度的GO干预H9c2细胞12h,MTT比色法检测H9c2细胞的存活率;光学显微镜下观察H9c2细胞形态学；AnnexinV/PI结合流式细胞仪检测凋亡率；2’,7’-二氯荧光黄双乙酸盐（DCFH-DA）探针检测细胞内活性氧簇（reactive oxygen species,ROS）水平.结果 （1）随G/GO浓度增加,H9c2细胞的存活率逐渐降低,其中1.6 mU/mL、2 mU/mL、2.4 mU/mL和2.8 mU/mL组存活率显著低于对照组,差异有统计学意义（P＜0.05）；其中G（33 mmol/L）/GO（2 mU/mL）的存活率为49.3％±6.7％.（2）随G/GO浓度增加,H9c2细胞的细胞凋亡率逐渐升高,细胞呈现典型的凋亡特征,其中1.6 m U/mL、2 mU/mL、2.4 mU/mL和2.8 mU/mL凋亡率显著高于对照组,差异有统计学意义（P＜0.05）；其中G（33 mmol/L）/GO（2 mU/mL）的细胞凋亡率为44.56％±3.02％.（3）G（33 mmol/L）/GO（2 mU/mL）作用H9c2细胞12h时,ROS含量显著高于对照组,差异有统计学意义（荧光强度：47.70％±2.09％ vs.17.59％±0.75％,P＜0.05）.结论 G/GO引起的H9c2细胞氧化应激所致的凋亡有剂量依赖性,G（33 mmol/L）/GO（2 mU/mL）是建立H9c2心肌细胞凋亡模型的合适浓度.

Establishment of models of cardiomyocyte apoptosis in H9c2 cells induced by glucose/glucose oxidase

Objectives To investigate the effects of different concentrations of glucose （G）/glucose oxidase （GO） on the apoptosis of H9c2 cells,and to establish a model for oxidative stress-induced apoptosis in H9c2 cells.Methods The H9c2 cells were treated with 33 mmol/L glucose and serial concentrations of GO for 12 h.The viability of H9c2 cells was evaluated by MTT assay ; the morphology of H9c2 cells was observed under microscope ; the apoptosis was measured by flow cytometry and Annexin V/PI ; the generation of reactive oxygen species （ROS-superoxide anion and hydrogen peroxide） was measured by DCFH-DA assay.Results （1） The viability of H9c2 cells decreased with the increase of GO concentration,and those in 1.6 mU/mL group,2 mU/mL group,2.4 mU/mL group and 2.8 mU/mL group were significantly lower than that in control group （P〈0.05）,among which,that of G （33 mmol/L） / GO （2 mU/mL） was 49.3%±6.7%.（2） The apoptotic rate increased with the increase of G/GO concentration,and those in 1.6 mU/mL group,2 mU/mL group,2.4 mU/mL group and 2.8 mU/mL group were significantly higher than that in control group （P〈0.05）,among which,that of G （33 mmol/L） / GO （2 mU/mL） was 44.56%±3.02%.（3） As compared with that in control group,the level of ROS significantly increased 12 h after being treated with G （33 mmol/L）/GO （2 mU/mL） （fluorescence intensity：47.70%±2.09% vs.17.59% ± 0.75%,P〈0.05）.Conclusions The oxidative stress induced apoptosis caused by G/GO in H9c2 cells is dose-dependent,and G （33 mmol/L） / GO （2 mU/mL） may be the ideal concentration to establish the apoptosis model in H9c2 cells.