Cameleon测钙系统在H_2O_2诱导的A549细胞凋亡过程中的应用

Application of the Cameleon Monitoring System of Ca^(2+) in the Process of H_2O_2 Induced Apoptosis of A549 Cells

观察Cameleon测钙系统在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),并探讨[Ca2+]i与细胞凋亡和Pyk2磷酸化的关系。采用Ca2+指示器Cameleon YC3.6转染A549细胞,24 h后用50 mmol/L H2O2刺激细胞,激光扫描共聚集显微镜实时测定选取细胞的[Ca2+]i变化。采用Western blot检测H2O2刺激的细胞中Pyk2-tyr402磷酸化水平。采用DAPI染色试剂盒观察H2O2刺激后细胞凋亡情况。结果发现,在H2O2作用下,A549细胞胞浆内游离[Ca2+]i迅速升高,同时Pyk2-tyr402磷酸化水平显著升高(P<0.05),凋亡细胞百分比显著增加(P<0.01)。因此,H2O2促进A549细胞内Ca2+释放,可能通过活化Pyk2诱导细胞凋亡。

The aim was to evaluate the Cameleon monitoring system of Ca2~ in the process of H202 induced the apoptosis of lung adenocarcinoma A549 cells. The cytoplastic Ca2＋ concentration （[Ca：＋]i） was determined in real-time, and the correlations between [Ca2＋]i and cell apoptosis as well as proline-rich tyrosine kinase 2 （Pyk2） phosphorylation were explored. The Ca2＋ indicator Cameleon YC3.6 was transfected into A549 cells, and cells were stimulated with 50 mmol/L H2O2 24 h later. Laser scanning confocal microscope was applied to perform real-time monitoring on the variation of [Ca2＋]i in selected cells. Western blot assay was used to evaluate the activation of Pyk2-tyr402, and DAPI staining was used to observe apoptosis in H202 treated cells. Our results showed that the cytoplastic free [Ca2＋]i was rapidly elevated in H2Oz stimulated A549 cells. Pyk2-tyr402 was significantly phospho- rylated, and the apoptotic rate was increased in H202 treated cells, compared with untreated ones （P〈0.01）. In sum- mary, H202 promoted Ca2＋ release in A549 cells, and induced cell aoootosis Probablv bv activating Pvk2.