基于CFTR的胞浆内第二信使cAMP检测方法的建立

Establishment of a CFTR-based detection method for the second messenger cAMP in the cytoplasm

目的:建立一种基于CFTR可敏感检测胞浆内第二信使cAMP的检测方法。方法:构建CFTR和YFP-H148Q/I152L真核表达载体,应用脂质体转染法构建共表达CFTR和YFP-H148Q/I152L的FRT细胞,倒置荧光显微镜观察其表达情况,流式细胞仪检测细胞纯度;荧光淬灭动力学实验验证细胞模型的有效性;荧光淬灭动力学实验验证细胞模型可筛选CFTR调节剂;放射免疫法检测加入CFTR激活剂后细胞内的cAMP浓度。结果:倒置荧光显微镜下观察到CFTR表达在细胞膜上,YFP-H148Q/I152L表达于胞浆中;成功构建共表达CFTR和YFP-H148Q/I152L的FRT细胞模型;荧光变化斜率值与CFTR调节剂浓度成剂量依赖关系,该模型可筛选CFTR调节剂;荧光变化斜率值可反映胞浆内cAMP浓度,该模型可敏感检测胞浆内cAMP浓度。结论:此细胞模型可以高效敏感检测胞浆内第二信使cAMP浓度,为cAMP信号相关靶点的研究提供一种简便快捷的方法。

Objective:To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator(CFTR)that can sensitively detect the second messenger cyclic AMP(cAMP)in the cytoplasm.Methods:The eukaryotic expression vectors of CFTR and YFP-H148Q/I152L were constructed respectively.FRT cells co-expressing CFTR and YFP-H148Q/I152L were obtained by liposome transfection.The expression of CFTR and YFP-H148Q/I152L in FRT cells was observed by an inverted fluorescence microscopy,and flow cytometry was used to detect the purity of cells;The cell model was identified by the fluorescence quenching kinetics test.The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments.The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator.Results:The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm of FRT cells.The FRT cell model stably co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed.The model could screen CFTR modulators,and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner.The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm.The cell model could sensitively detect the intracellular cAMP concentration.Conclusion:The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm,and it provided a simple and efficient method for the study of other targets associated cAMP signal.