大鼠心肌细胞H9C2活性氧水平检测方法探究

Investigation of detection method of ROS in H9C2 cells

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（P <0.05），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 different methods for ROS measurement, and analyze their advantages and shortcomings so as to select a suitable and accurate one for detecting ROS in hypoxia-reoxygenation (H-R) model of rat myocardial cell line H9C2. Methods H9C2 cells were plated in an appropriate confluence (80%-90%).The experimental group (H-R 6 h) was exposed to hypoxia for 6 h followed by reoxygenation for 2 h, the control group (CON) was cultured in normoxic condition. Then DCFH-DA fluorescent probe was added into H9C2 cells.After incubation, the fluorescent intensity was detected by fluorescent microscopy, flow cytometer and fluorescence multiscan. Then these data were analyzed with appropriate analysis software. Results Compared with the control group, the results of flow cytometer [CON (152,690 ±34,104) FU/μg, H-R 6 h (325,669 ±12,755) FU/μg] or fluorescence multiscan [CON (282.3 ±12.57) FU/μg, H-R 6 h (1,274 ±37.05) FU/μg] showed that the ROS level of the experimental group was significantly higher than that of the control group (P < 0.05), but the results from fluorescence microscopy could not distinguish the experimental group from the control group. Conclusions Fluorescence multiscan can better determine the ROS level in H9C2 cells.