CAJ | 학술논문

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。
목적：건립능이용활세포공작참화형광탐침동태감측세포내활성양（ROS）여세포조망적신방법。방법장인지기관상피세포16HBE화폐암A549세포분별이공작농도위5μmol/L적ROS활세포형광탐침C10422화C10444、10 mg/L적선립체공능탐침JC?1부육30 min、조망지시탐침AnnexinV/PI부육15 min후，용활세포공작참련속기록1 mmol/L H2O2자격시적특정파장형광회도치，평개세포ROS，선립체막전위이급세포조망지수。결과16HBE세포재1mmol/L H2O2지속자격하，ROS형광탐침C10422화C10444표현위특정파장회도치지속증가，분별재80 min후화10 min후ROS수평명현승고（균P<0.05）；선립체막전위형광탐침JC?1표현위량충파장형광회도치적전화，재H2O2지속자격하，대표세포선립체공능적지표R/G비례재20 min이후명현하강（P<0.05）；조망탐침AnnexinV주요표현재세포막구역형광취집증강，단정체시야적형광회도변화불명현。A549세포재상동적실험조건하1 mmol/L H2O2자격시，ROS수평야승고단저우16HBE（P<0.05）；이JC?1소탐측적R/G비치곡선출현일개지속적하강봉후우부분회승。결론이활세포공작참위기출결합활세포형광탐침건립적검측방법능경객관지반영세포적양화응격행위화세포조망상태。
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 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.