大鼠心肌细胞H9C2活性氧水平检测方法探究

发布时间：2018-10-15 14:27

【摘要】：目的比较3种细胞内活性氧(ROS)的检测方法,分析优缺点,筛选1种合适、准确的检测大鼠心肌细胞H9C2缺氧/复氧(H/R)模型中细胞内活性氧的方法。方法培养H9C2细胞,选择密度合适(80%~90%)的H9C2细胞进行铺板,加入2,7-双乙酸二氯荧光素(DCFH-DA)荧光探针孵育,应用荧光显微镜、流式细胞仪及荧光酶标仪3种方法检测对照组(正常含氧量培养条件,CON)和实验组(缺氧复氧处理,H/R6h)心肌细胞的荧光强度,从而反映大鼠心肌细胞内活性氧水平,并利用数据分析软件进行统计分析。结果流式细胞仪检测分析结果:对照组(152 690±34 104)FU/μg,实验组(325 669±12 755)FU/μg;荧光酶标仪检测分析结果:对照组(282.3±12.57)FU/μg,实验组(1 274±37.05)FU/μg。实验组的ROS水平与对照组的ROS水平比较,差异有统计学意义(P0.05),实验组高于对照组,而荧光显微镜观察法无法定量地分析出实验结果。结论 3种ROS检测方法中,荧光酶标仪检测方法能更好地判断大鼠心肌细胞H9C2活性氧水平。
[Abstract]:Objective to compare three methods for the detection of intracellular reactive oxygen species (ROS), analyze their advantages and disadvantages, and select a suitable and accurate method for the detection of intracellular reactive oxygen species (Ros) in rat cardiomyocytes with hypoxia / reoxygenation (H / R) model. Methods H9C2 cells were cultured, and H9C2 cells with suitable density (80%) were selected and incubated with fluorescein dichloroacetate (DCFH-DA) probe. The cells were incubated with fluorescence microscope. Flow cytometry (FCM) and fluorescence enzyme labeling (FCM) were used to detect the fluorescence intensity of cardiac myocytes in control group (normal oxygen content culture condition, CON) and experimental group (hypoxia reoxygenation, H/R6h), so as to reflect the level of reactive oxygen species (Ros) in rat cardiomyocytes. And use data analysis software for statistical analysis. Results the results of flow cytometry were (152,690 卤34,104) FU/ 渭 g in the control group and (325,669 卤12,755) FU/ 渭 g in the experimental group, and the results of fluorescence enzyme labeling were (282.3 卤12.57) FU/ 渭 g in the control group and (1 274 卤37.05) FU/ 渭 g in the experimental group. The level of ROS in the experimental group was significantly higher than that in the control group (P0.05) compared with the ROS level of the control group, but the fluorescence microscope could not quantitatively analyze the results of the experiment. Conclusion among the three ROS detection methods, the fluorescent enzyme labeling method can better judge the level of H9C2 reactive oxygen species in rat cardiomyocytes.
【作者单位】：四川大学华西医院公共实验技术中心;
【分类号】：R-332

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