应用Ca^(2+)荧光探针fluo-3和fluo-4测定H_2O_2诱导的A549细胞凋亡过程中的[Ca^(2+)]_i变化  

Application of the Ca^(2+) Indicator Fluo-3 and Fluo-4 in the Process of H_2O_2 Induced Apoptosis of A549 Cell

作　　者：张四洋[1] 李春艳[1] 高建[1] 邱雪杉[2] 崔泽实[1] 

机构地区：[1]中国医科大学实验技术中心,沈阳110001 [2]中国医科大学病理学教研室,沈阳110001

出　　处：《中国肺癌杂志》2014年第3期197-202,共6页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金项目(No.81101599);沈阳市科学技术项目(No.F11-241-00,No.F12-264-4-01);辽宁省科学事业研究公益基金项目(No.2012006005)资助~~

摘　　要：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fluo-4探针负载的选定细胞中[Ca2+]i变化范围是7.6nM-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fluo-4可能更适合于监测含量较低的细胞中[Ca2+]i变化。Background and objective Lung cancer is a common malignant tumor all over the world, and Ca^2＋ is a critical regulator for apoptosis of cancer cells. The monitoring of cytoplastic Ca^2＋ level in real-time will contribute to further investigate the molecular mechanisms of apoptosis mediated by Ca^2＋ in lung cancer cells. To evaluate the Ca^2＋ indicator fluo-3 and fluo-4 in the process of H2O2 induced the apoptosis of lung adenocarcinoma A549 cells. The cytoplastic Ca^2＋ concentration （[Ca^2＋]i,） was determined in real-time, and the correlations between [Ca^2＋]i and cell apoptosis were investigated. The differences in fluorescence intensity and measured value were compared between the two Ca^2＋ indicators. Methods Cells were loaded with the Ca^2＋ indicator fluo-3 or fluo-4 for 1 h, and then stimulated with S0 mM H2O2. Laser scanning confocal microscope was applied to perform real-time monitoring on the variation of [Ca^2＋]i in selected cells. DAPI staining was used to observe apoptosis in H2O2 treated cells. Results Our results showed that the fluorescence intensity offluo-4 was stronger than that offluo-3 in the same condition of dye concentration, loading time and image acquisition parameters before or after H202 stimulation. The cytoplastic [Ca^2＋], was rapidly elevated in H2O2 stimulated A549 cells. The range of [Ca^2＋]i, in selected cells loaded with fluo-3 was 112.2 nM-1,069.6 nM, and that in selected cells loaded with fluo-4 was 7.6 nM-505.4 nM. Moreover, the apoptotic rate was significantly increased in H2O2 treated cells, compared with untreated ones （P〈0.01）. Conclusion In summary, H2O2 promoted Ca^2＋ release in A549 cells, and induced cell apoptosis. Ca^2＋ indicator fluo-4 was probably more applicable to measure [Ca^2＋]i in cells with less content of Ca^2＋.

关 键 词：Fluo-3 Fluo-4 CA^2+ H2O2 细胞凋亡 

分 类 号：R734.2[医药卫生—肿瘤]