基于CFTR的H2受体调节剂高通量筛选细胞模型的构建

Construction of High Throughput Screening Cell Model for H2 Receptor Modulators Based on CFTR

目的建立基于囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator, CFTR)高通量筛选组胺H2受体(histocompatibility-2)调节剂的模型。方法应用RT-PCR、Western blot检测H2受体在Fischer大鼠甲状腺滤泡上皮(FRT)细胞中表达情况。采用脂质体转染获取同时表达CFTR和黄色荧光蛋白双突变体YFP-H148Q/I152L的FRT细胞。荧光淬灭动力学实验检测细胞模型功能,判断该模型是否可以筛选H2受体的调节剂。结果 RT-PCR、Western blot结果显示,FRT内源性表达H2受体。采用倒置荧光显微镜观察,证实成功构建同时表达CFTR和YFP-H148Q/I152L的细胞模型。荧光淬灭动力学实验证明该模型具有cAMP激活氯离子通道的特性,可用于H2受体调节剂的筛选,且荧光斜率(slope)值与H2受体调节剂的浓度呈剂量依赖关系。结论成功构建基于CFTR高通量筛选H2受体调节剂的细胞模型。

OBJECTIVE To establish a high-throughput screening model of histamine H2 receptor regulators based on cystic fibrosis transmembrane conduction regulator(CFTR). METHODS RT-PCR and Western blot were used to detect the expression of H2 receptor in thyroid follicular epithelial(FRT) cells of Fischer rats. FRT cells expressing CFTR and YFP-H148 Q/I152 L were transfected with liposomes. Fluorescence quenching kinetics test was used to detect the function of the cell model to determine whether the model could screen regulators of H2 receptor. RESULTS RT-PCR and Western blot showed that FRT endogenous expressed H2 receptor. Fluorescence microscopy confirmed that CFTR and YFP-H148 Q/I152 L cell models were successfully constructed. The fluorescence quenching kinetics experiments showed that the model had the characteristics of cAMP activated chloride channels, and could be used to screen H2 receptor modulators. The slope of fluorescence was dose-dependent with the concentration of H2 receptor modulators. CONCLUSION The high-throughput screening cell model of H2 receptor modulators based on CFTR is successfully constructed.