Background and objective Lung cancer is a common malignant tumor all over the world, and Ca2+is a critical regulator for apoptosis of cancer cells. hTe monitoring of cytoplastic Ca2+level in real-time will contribute to further investigate the molecular mechanisms of apoptosis mediated by Ca2+in lung cancer cells. To evaluate the Ca2+indicator lfuo-3 and lfuo-4 in the process of H2O2 induced the apoptosis of lung adenocarcinoma A549 cells. hTe cytoplastic Ca2+concentration ([Ca2+]i) was determined in real-time, and the correlations between [Ca2+]i and cell apoptosis were investigated. hTe differences in lfuorescence intensity and measured value were compared between the two Ca2+indicators. Methods Cells were loaded with the Ca2+indicator lfuo-3 or lfuo-4 for 1 h, and then stimulated with 50 mM H2O2. Laser scanning confocal microscope was applied to perform real-time monitoring on the variation of [Ca2+]i in selected cells. DAPI staining was used to observe apop-tosis in H2O2 treated cells. Results Our results showed that the lfuorescence intensity of lfuo-4 was stronger than that of lfuo-3 in the same condition of dye concentration, loading time and image acquisition parameters before or atfer H2O2 stimulation. hTe cytoplastic [Ca2+]i was rapidly elevated in H2O2 stimulated A549 cells. hTe range of [Ca2+]i in selected cells loaded with lfuo-3 was 112.2 nM-1,069.6 nM, and that in selected cells loaded with lfuo-4 was 7.6 nM-505.4 nM. Moreover, the apoptotic rate was signiifcantly increased in H2O2 treated cells, compared with untreated ones (P<0.01). Conclusion In summary, H2O2 promoted Ca2+release in A549 cells, and induced cell apoptosis. Ca2+indicator lfuo-4 was probably more applicable to measure [Ca2+]i in cells with less content of Ca2+.%背景与目的肺癌是世界范围内常见的恶性肿瘤，Ca2+对于肿瘤细胞凋亡有重要的调控作用。实时监测肺癌细胞内Ca2+水平，有助于深入研究Ca2+介导肺癌细胞凋亡的分子机制。本研究旨在观察Ca2+荧光探针fluo-3和fluo-4在H2O2诱导的A549细胞凋亡过程中的应用，实时测定胞浆Ca2+浓度（[Ca2+]i），探讨[Ca2+]i与细胞凋亡的关系，并比较两种Ca2+探针在荧光强度及[Ca2+]i测定值方面的差异。方法采用Ca2+荧光探针fluo-3和fluo-4负载细胞，1 h后用不同浓度的H2O2刺激细胞，激光扫描共聚焦显微镜实时测定选取细胞的[Ca2+]i变化。采用DAPI染色试剂盒观察H2O2刺激后细胞凋亡情况。结果在相同的探针浓度、负载时间和相同的图像采集参数的条件下，选定细胞内fluo-4平均荧光强度高于fluo-3。50 mM H2O2刺激后，A549细胞胞浆内[Ca2+]i迅速升高，通过公式计算发现采用fluo-3探针负载的选定细胞中[Ca2+]i变化范围是112.2 nM-1,069.6 nM，采用lfuo-4探针负载的选定细胞中[Ca2+]i变化范围是7.6 nM-505.4 nM。同时发现经H2O2刺激后，凋亡细胞百分比明显增加（P<0.01）。结论H2O2促进A549细胞内Ca2+释放，诱导细胞凋亡。Ca2+探针lfuo-4可能更适合于监测含量较低的细胞中[Ca2+]i变化。
展开▼
