高通量筛选瞬时受体势V3通道调节剂细胞模型的建立

Establishment of a Cell Based High Throughput Screening Model for Transient Receptor Potential V3 Modulators

摘要:

为发现TRPV3调节剂, 通过荧光成像分析系统检测钙浓度, 建立高通量筛选瞬时受体势V3通道(Transient receptor potential V3, TRPV3)调节剂的细胞模型。将TRPV3表达载体转染人胚肾(HEK-293)细胞, 抗生素筛选稳定表达TRPV3的细胞系, 选取TRPV3特异性调节剂作用于细胞模型, 应用荧光成像分析系统测钙实验检测TRPV3高表达细胞系药理学特征, 同时优化实验条件, 考察模型的稳定性, 并评估应用于96孔板及384孔板进行高通量筛选的可靠性及准确性。获得了高表达TRPV3的HEK-293稳定细胞系, 通过TRPV3离子通道调节的钙流信号与TRPV3特异性调节剂成剂量依赖关系, 优化得到了最适筛选条件, 该模型稳定, 灵敏, 通过Z因子及Spiking检测, 完全符合高通量筛选需求。利用此细胞模型通过检测钙信号可筛选TRPV3调节剂。

Abstract:

We established a cell based high throughput screening model by calcium assay on fluorometric imaging plate reader for finding modulators of TRPV3．The TRPV3 expression vector was transfected into HEK-293 and table cell line expressing TRPV3 was selected with antibiotics. Upon TRPV3 specific modulators stimulated, pharmacological characteristics of TRPV3 over expression cell line were detected by calcium assay on fluorometric imaging plate reader. Assay conditions were optimized and stability of the model was observed. The reliability and accuracy of application to 96 and 384 well format high throughput screening were also evaluated. A stable HEK-293 cell line highly expressing TRPV3 was established. TRPV3 specific modulators could modulate calcium signal through TRPV3 in dose dependent manner. Optimized screening condition was established by assay development. This model is stable and sensitive, and meets the requirement of high throughput screening by Z'factor validation and Spiking test. This cell model can be applied to screening TRPV3 modulators by calcium assay.