摘要
目的建立一种基于钙激活氯离子通道(calcium-activated chloride channel,CaCC)靶向M3受体(cholinergic receptor muscarinic 3,Chrm3)高效敏感的药物细胞筛选模型和方法。方法RT-PCR、免疫荧光技术及Western blot检测Chrm3在FRT细胞中的表达情况;构建共表达钙激活氯离子通道蛋白1(anoctamin 1,ANO1)和YFP-H148Q/I152L的Fischer大鼠甲状腺滤泡上皮(Fischer rat thyroid,FRT)细胞模型,倒置荧光显微镜和荧光淬灭动力学实验鉴定CaCC细胞模型有效性;Fura-2荧光探针法检测加入Chrm3激活剂后胞浆内的钙浓度,荧光淬灭动力学实验验证细胞模型可筛选Chrm3调节剂;Z'因子评估该细胞模型是否适用于高通量筛选。结果FRT细胞在mRNA及蛋白水平均内源性表达Chrm3;成功构建了稳定共表达ANO1和YFP-H148Q/I152L的FRT细胞模型;该细胞模型可敏感检测细胞内钙浓度,相对荧光强度变化值与Chrm3调节剂浓度成剂量依赖关系,可用于Chrm3调节剂的筛选;该模型的Z'因子值为0.678,满足高通量筛选的要求。结论此细胞模型通过敏感检测钙信号实现了对Chrm3调节剂的高通量筛选,也可应用于对其他与钙离子信号相关G蛋白偶联受体(G-protein-coupled receptor,GPCR)靶点的筛选。
OBJECTIVE To establish an efficient and sensitive drug cell screening model and method targeting Chrm3 based on calcium-activated chloride channel(CaCC).METHODS RT-PCR,immunofluorescence and Western blot were applied to detect the expression of Chrm3 in FRT cells.The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was constructed,and the CaCC cell model was identified by an inverted fluorescence microscope and fluorescence quenching kinetics test.The Fura-2 fluorescent probe was used to detect the calcium concentration in cytoplasm after adding Chrm3 activator.The validation of the cell model which could screen Chrm3 modulators was verified by the fluorescence quenching kinetics experiments.Z'factor was calculated to evaluate the sensitivity and specificity of the cell model.RESULTS FRT cells endogenously expressed Chrm3.The FRT cell model stably co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed.The cell model could sensitively detect intracellular calcium concentration.The value of relative fluorescence intensity changed with the concentration of Chrm3 modulators in a dose-dependent manner,and the model could screen Chrm3 modulators.The Z'factor was 0.678,which met the requirement for high-throughput screening.CONCLUSION The cell model can be applied for high-throughput screening Chrm3 modulators,and it can sensitively detect calcium signals.The cell model can also screen other GPCR regulators-associated calcium signaling.
作者
肖云萍
解宇浩
张嘉琪
郝峰
王国庆
XIAO Yun-ping;XIE Yu-hao;ZHANG Jia-qi;HAO Feng;WANG Guo-qing(School of Laboratory Medical,Beihua University,Jilin 132013,China;Laboratory Medical College,Jilin Medical College,Jilin 132013,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2021年第9期715-722,共8页
Chinese Pharmaceutical Journal
基金
国家自然科学基金项目资助(81601234)
吉林省大学生创新创业训练计划资助(201913706071
202013706024)
吉林省中医药科技项目(2021092)。
关键词
M_(3)受体
钙激活氯离子通道
细胞模型
高通量筛选
钙浓度
cholinergic receptor muscarinic 3
calcium-activated chloride channel
cell model
high-throughput screening
calcium concentration
