肥大细胞抗原活化时钙离子信号变化的研究

目的为哮喘等Ⅰ型变态反应性疾病的靶向信号转导分子治疗寻找药物设计新靶点提供基础。方法应用钙离子荧光指示剂Fluo-3，通过流式细胞仪和激光共聚焦荧光显微镜分析肥大细胞RBL-2H3活化时细胞内钙离子浓度（［Ca2＋］i）的变化和细胞外钙离子浓度对释放组胺的影响，观察钙离子信号与肥大细胞活化的关系。结果抗原可使致敏RBL-2H3细胞迅速释放组胺和升高［Ca2＋］i，90~110s时最强达（525±42）nmol／L（n＝12），激光共聚焦荧光显微镜观察到胞浆内充满绿荧光；胞外加入钙离子螫合剂EDTA后［ca2＋］i为（80±4）nmol／L，与基础［Ca2＋］i（79±3）nmol／L差异无显著性（P＞0．05，n＝12）；但显著增高磷酸酪氨酸浓度；加入佛波酯后再加入DNP－BSA，前后［Ca2＋］i差异无显著性（P＞0．05，n＝8）。结论　钙离子参与致敏肥大细胞的抗原活化过程。

A study on the changes of calcium signal in mast cells activated by antigen

OBJECTIVE: To study the relationship between the calcium signal and activation of mast cells, laying the foundation for discovering a new potential theraputical target of signal transduction for type I anaphylactic diseases like asthma. METHODS: The fluorescent Ca2+ indicator Fluo-3 was used to observe and quantitate the calcium signal directly in activated RBL-2H3 mast cells by a flow cytometer and a confocal fluorescence microscope with laser. RESULTS: Antigens elicited significant increase in intracellular Ca2+ concentration (Ca2+]i) and histamine release in sensitized cells, maximal Ca2+]i increase being reached (525 +/- 42) nmol/L (n = 12) within 90-110 s after stimulation. Increase of fluo-3-fluorescence intensity was also observed with the confocal fluorescence microscope in cytosol. No significant increase of Ca2+]i was observed after addition of EDTA in sensitized cells from an initial value of (79 +/- 3) nmol/L, to (80 +/- 4) nmol/L (P > 0.05, n = 12), but tyrosine phosphorylation was readily observed, and the indirect fluorescent intensity was significantly greater than that in the control group. No significant change of Ca2+]i was observed with the addition of DNP-BSA after PMA (P > 0.05, n = 8). CONCLUSION: Calcium is involved in the activation process of mast cells stimulated by antigens.