Ca^(2+)荧光蛋白探针Cameleon和荧光染料探针fluo-3测定H_2O_2刺激的A549细胞中Ca^(2+)浓度变化

Application of Ca^(2+) indicators of the fluorescent protein Cameleon and the fluorescent dye fluo-3 in H_2O_2stimulated A549 cells

目的:本研究旨在观察Ca2+荧光蛋白探针Cameleon和荧光染料探针fluo-3在H2O2刺激的A549细胞中的应用,实时测定细胞质Ca2+浓度([Ca2+]i),并比较两种Ca2+探针在荧光曲线及[Ca2+]i测定值等方面的差异。方法:采用Ca2+荧光蛋白探针Cameleon YC3.6转染A549细胞,24h后用50mmol/L 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Ca2+荧光染料探针fluo-3负载细胞,1h后用50mmol/L 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:采用Cameleon探针转染的细胞中,反映[Ca2+]i变化的R值曲线迅速升高后逐渐降至刺激前水平,通过公式计算发现[Ca2+]i变化范围是33.1-596.1 nmol/L。采用fluo-3探针负载的细胞中,反映[Ca2+]i变化的荧光强度F值曲线逐渐升高至稳定,通过公式计算得到[Ca2+]i变化范围是131.8-695.6 nmol/L。同时发现经H2O2刺激后,凋亡细胞百分比显著增加(P<0.01)。结论:Ca2+荧光蛋白探针Cameleon YC3.6和荧光染料探针fluo-3均检测到H2O2诱导的细胞内[Ca2+]i变化,Cameleon YC3.6可能更灵敏地反映快速变化的钙信号。

Objective：To evaluate the Cameleon and fluo 3 monitoring system of Ca2＋ in H2O2 stimulated lung adenocarcinoma A549 cells. The cytoplastic Ca2 ＋ concentration （ [ Ca2 ＋ ] i ） was determined by real time and the differences in fluorescence curves and measured value were compared between the two Ca2 ＋ indicators. Methods：The Ca2＋ indicator Cameleon YC3.6 was transfected into A549 cells, and cells were stimulated with 50mmol/L H2 O2 24h later. Cells were loaded with the Ca2＋ indicator fluo 3 for 1 h, and then stimulated with 50mmol/L H2 O2. Laser scan ning confocal microscope was applied to perform real time monitoring on the variation of [ Ca2 ~ ] i in selected cells. DAPI staining was used to confirm apoptosis in H2 O2 treated cells. Results：The R curves reflecting [ Ca2＋ ] i in cells transfected with Cameleon YC3.6 were elevated rapidly to the peak then lowered gradually to the level before stimula ting, and the range of [ Ca2 ＋ ] i was 33.1 596.1 nmol/L. The F curves representing [ Ca2 ＋ ] i in cells loaded with fluo 3 were gradually elevated to a stable level, and the range of [ Ca2 ＋ ] i was 131.8 695.6 nmol/L. Moreover, the apoptotic rate was increased in H2 02 treated cells, compared with untreated ones （P 〈 0.01 ）. Conclusion： [ Ca2 ＋ ] i was determined in H2 02 stimulated A549 cells by Ca2 ＋ indicators of both the fluorescent protein Cameleon YC3.6 and the fluorescent dye fluo 3, and Cameleon YC3.6 was probably more sensitive to capture the rapid variation of calci um signals.