基于CaCC的TRPV4调节剂高通量筛选细胞模型的建立  

Establishment of a cell model for high-throughput TRPV4 regulator screening based on CaCC

作　　者：肖云萍 解宇浩 张嘉琪 洪啟元 郝峰 王国庆 Xiao Yun-Ping;Xie Yu-Hao;Zhang Jia-Qi;Hong Qi-Yuan;Hao Feng;Wang Guo-Qing(Laboratory Medical College,Jilin Medical College,Jilin City,Jilin 132013,China;School of Laboratory Medical,Beihua University,Jilin City,Jilin 132013,China;School of Public Health,Jilin Medical College,Jilin City,Jilin 132013,China)

机构地区：[1]吉林医药学院检验学院,吉林省吉林市132013 [2]北华大学医学技术学院,吉林省吉林市132013 [3]吉林医药学院公共卫生学院,吉林省吉林市132013

出　　处：《解放军医学杂志》2020年第10期1032-1039,共8页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81601234);吉林省教育厅基金(JJKH20170418KJ,JJKH20191056KJ);吉林省卫生与健康技术创新项目(2018J113);吉林省大学生创新创业训练计划(201913706071)。

摘　　要：目的构建基于钙激活氯离子通道(CaCC)的瞬时受体电位香草酸亚型4(TRPV4)调节剂的高通量筛选细胞模型。方法采用RT-PCR检测Fischer大鼠甲状腺滤泡上皮(FRT)细胞中内源性表达的TRPV4,所得PCR产物切胶回收后测序;采用Western blotting检测FRT细胞中TRPV4蛋白的表达情况。应用脂质体转染法构建共表达钙激活氯离子通道蛋白1(ANO1)和YFP-H148Q/I152L的FRT细胞模型,倒置荧光显微镜下观察ANO1和YFP-H148Q/I152L在细胞中的表达情况,应用荧光淬灭动力学实验测定细胞模型的有效性。加入TRPV4激活剂和抑制剂,应用荧光淬灭动力学实验检测模型能否筛选TRPV4调节剂;加入TRPV4激活剂,应用Fura-2荧光探针法检测细胞内的Ca2+浓度;通过Z'因子评估细胞模型是否适用于高通量筛选。结果RT-PCR和Western blotting检测结果显示,FRT细胞内源性表达TRPV4。倒置荧光显微镜下清晰可见ANO1表达在FRT细胞膜上,YFP-H148Q/I152L表达在FRT细胞质中,即成功构建了共表达ANO1和YFP-H148Q/I152L的FRT细胞模型。荧光淬灭动力学实验结果显示,该模型可以筛选TRPV4调节剂,斜率(Slope)值与TRPV4调节剂的浓度呈剂量依赖关系;该模型可敏感检测细胞内Ca2+的浓度变化,通过Slope值可反映细胞内Ca2+浓度的高低;Z'因子为0.728,表明该模型可高通量筛选TRPV4调节剂。结论成功构建了一种可以敏感高效筛选TRPV4调节剂的高通量筛选细胞模型。Objective To construct a high-throughput screening model for transient receptor potential vanilloid 4(TRPV4)channel modulators based on calcium-activated chloride channels(CaCC).Methods RT-PCR was used to detect the endogenous expression of TRPV4 in Fischer rat thyroid(FRT)cells.The PCR products obtained were subjected to nucleic acid sequencing using gel-recovery technology.Western blotting was employed to detect the expression of TRPV4 protein in FRT cells.The liposome transfection method was applied to construct the FRT cell model that co-expressed anoctamin 1(ANO1)and YFP-H148Q/I152L.The expressions of ANO1 and YFP-H148Q/I152L in cells were identified by the inverted fluorescence microscope and the fluorescence quenching kinetics test.After adding TRPV4 activators and inhibitors,the fluorescence quenching kinetics experiment was used to test whether the model could screen TRPV4 modulators.The Fura-2 fluorescent probe method was applied to detect the calcium concentration in cells after adding TRPV4 activators;The Z'factor was calculated to evaluate the sensitivity and specificity of the cell model.Results RT-PCR and Western blotting confirmed the endogenous expression of TRPV4 in FRT cells;ANO1 was clearly expressed on the FRT cell membrane and YFP-H148Q/I152L was clearly expressed in the cytoplasm of FRT cells under the inverted fluorescence microscope.The FRT cell model co-expressing ANO1 and YFP-H148Q/I152L was successfully constructed.Fluorescence quenching kinetics experiments confirmed that the model could screen TRPV4 regulators,and the slope value of fluorescence change and the concentration of TRPV4 regulator concentration were in a dose-dependent manner.The model could sensitively detect changes in intracellular calcium concentration,and the slope value could reflect intracellular calcium concentration.The Z'factor was 0.728,which demonstrates its capacity for high-throughput screening.Conclusions We successfully constructed a high-throughput model that could screen TRPV4 modulators sensitively and

关 键 词：瞬时受体电位香草酸亚型4 钙激活氯离子通道 YFP-H148Q/I152L 高通量筛选细胞模型 

分 类 号：R915[医药卫生—微生物与生化药学] R311[医药卫生—药学]