RBL-2H3细胞脱颗粒模型研究

Study on degranulation model of RBL-2H3 cells

RBL-2H3细胞是体外研究变态反应的常用细胞,为了建立RBL-2H3细胞脱颗粒检测模型,以C48/80作为阳性药物,分析比较了细胞形态观测法、台盼蓝染色法、β-氨基己糖苷酶释放率及钙离子质量浓度检测等细胞脱颗粒检测方法.通过对C48/80作用质量浓度及时间的摸索,建立C48/80刺激RBL-2H3细胞脱颗粒的最佳药物作用条件.结果表明,β-氨基己糖苷酶释放率与细胞脱颗粒程度存在显著量-效关系,且操作简单、结果稳定.C48/80工作质量浓度为60μg/mL,最佳作用时间40 min.在此基础上,优化了DNPIgE/DNP-BSA诱导细胞脱颗粒体系,0.4μg/mL DNP-IgE致敏RBL-2H3细胞过夜,10μg/mL DNP-BSA诱导细胞,细胞脱颗粒程度最严重.同时发现,不同释放介质对DNP-BSA诱导细胞脱颗粒有显著影响.该结果可为变态反应相关研究提供实验基础.

RBL-2H3 cells are commonly used to research on the pathogenesis of allergic diseases in vitro.In order to establish a detection model of degranulation of RBL-2H3 cells,C48/80 was used as a positive drug to analyze and compare different methods for cells degranulation detection,including the cell morphology observation,trypan blue staining,beta hexosaminidase release rate and calcium ion concentrations.Through studying the concentration and time of C48/80,the optimized drug condition was established for RBL-2H3 cell degranulation induced by C48/80.The results showed that the C48/80 concentration was 60μg/mL with 40 min,and there was dose-effect relationship between the degree of degranulation and the beta hexosaminidase release rate.Furthermore,this method was simple and reliable,the beta hexosaminidase release rate was applied to detect the degranulation of cells in subsequent experiments.when the model of cell degranulation was successfully induced by DNP-IgE/DNP-BSA,0.4μg/mL DNP-IgE sensitized RBL-2H3 cells overnight,and 10μg/mL DNP-BSA induced cells,the cell degranulation was the most serious.The results also showed that the cell degranulation was affected significantly by different release mediums.These results will provide the experimental basis for further studying the allergic diseases.