中文摘要：

目的：建立能利用活细胞工作站和荧光探针动态监测细胞内活性氧（ROS）与细胞凋亡的新方法。方法将人支气管上皮细胞16HBE和肺癌A549细胞分别以工作浓度为5μmol/L的ROS活细胞荧光探针C10422和C10444、10 mg/L的线粒体功能探针JC-1孵育30 min、凋亡指示探针AnnexinV/PI孵育15 min后，用活细胞工作站连续记录1 mmol/L H2O2刺激时的特定波长荧光灰度值，评价细胞ROS，线粒体膜电位以及细胞凋亡指数。结果16HBE细胞在1mmol/L H2O2持续刺激下，ROS荧光探针C10422和C10444表现为特定波长灰度值持续增加，分别在80 min后和10 min后ROS水平明显升高（均P〈0.05）；线粒体膜电位荧光探针JC-1表现为两种波长荧光灰度值的转化，在H2O2持续刺激下，代表细胞线粒体功能的指标R/G比例在20 min以后明显下降（P〈0.05）；凋亡探针AnnexinV主要表现在细胞膜区域荧光聚集增强，但整体视野的荧光灰度变化不明显。A549细胞在相同的实验条件下1 mmol/L H2O2刺激时，ROS水平也升高但低于16HBE（P〈0.05）；而JC-1所探测的R/G比值曲线出现一个持续的下降峰后又部分回升。结论以活细胞工作站为基础结合活细胞荧光探针建立的检测方法能更客观地反映细胞的氧化应激行为和细胞凋亡状态。

英文摘要：

Objective To established a new method of using living cell workstation and fluorescent probe for dynamic monitoring of intracellular reactive oxygen species（ROS）and cell apoptosis. Methods The cells were incubated in the working concentrations of 5 μmol/L ROS living cell fluorescent probes C10422 and C10444,and 10 mg/L mitochondrial membrane function probe JC-1 for 30 min,and in apoptosis indication probe AnnexinV/PI for 15 min. Then, the living cell workstation was used to continuously record the fluorescent grey value of specific wavelength during the 1 mmol/L H2O2 stimulation, and to evaluate the ROS level,mitochondrial membrane potential and apoptosis indexes. Results For the 16HBE and A549 cells,under the continuous stimulation of 1 mmol/L H2O2,the ROS living cell fluorescent probes C10422 and C10444 showed continuously increasing fluorescent grey value of specific wavelength, and the significantly increased ROS levels were reached at 80 min and 10 min,respectively（all P〈0.05）;the mitochondrial membrane function probe JC-1 showed the transition of the fluorescent grey value between the two specific wavelengths,and the significantly decreased R/G ratio,which was the index of cell mitochondrial function,was reached at 20 min（P〈0.05）;the apoptosis probe AnnexinV showed increased aggregation of fluorescence in the region of the cell membrane,but the fluorescent grey value of the general view was not significantly changed. For A549 cells,under the same experiment condition of 1mmol/L H2O2＆amp;nbsp;stimulation,the ROS level increased but was lower than that of 16HBE cells（P〈0.05）;the R/G ratio curve as detected with the mitochondrial membrane function probe JC-1 showed a continuous declining trough and then partially rebounced. Conclusion Living cell fluorescent probing based on living cell workstation can be more objective in reflecting the cell oxidative stress and apoptosis.