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为了建立高通量筛选TRPV3通道调节剂的方法,本研究利用FlexStation3酶标仪,针对瞬时转染TRPV3通道的HEK293T细胞的密度、Cal-520染料的浓度及染料孵育时间进行了条件优化.结果显示,40 000个细胞/孔、5.0％的Cal-520染料浓度及1.5 h的染料孵育时间为最佳实验条件.应用该筛选方法,筛选并发现来源于睡莲科植物的有效成分甲基莲心碱(neferine)能够抑制TRPV3通道,该抑制作用经全细胞膜片钳实验证实并测得其IC50为24.5±4.1 μM(n=6).对其它TRPV通道成员的选择性电生理学评价结果显示,甲基莲心碱对TRPVl和TRPV4通道无抑制作用.总之,实验数据显示,FlexStation3高通量钙荧光筛选可以用于发现TRPV3以及其他TRP通道的调节剂,筛选发现的天然产物甲基莲心碱可以作为TRPV3通道的工具药研究通道的药理学功能.“,”To develop a cell-based high-throughput calcium fluorescent assay for screening of TRPV3 channel modulators,we employed the FlexStation3 microplate reader and optimized conditions of the high-throughput screening (HTS) adaptable calcium fluorescent assay,such as cell density,incubation time and concentration of Cal-520 calcium fluorescent dye.The optimized FlexStation3 assay included the cell density of 40 000 cells per well,5.0％ of Cal-520 calcium fluorescent dye and 1.5 h of incubation time.Using the FlexStation3 assay in a 96-well format,we screened and identified a natural neferine from Nymphaeaceae plant that inhibited TRPV3 channel.To further confirm the inhibitory effect of neferine on TRPV3,we utilized the whole-cell patch clamp recordings and determined the dose-dependent inhibition of TRPV3 current by neferine with an IC50 at 24.5±4.1 μM (n =6) without inhibition of TRPV1 or TRPV4 channels.Taken together,our data showed that this validated HTS adaptable calcium fluorescent assay in FlexStation3 format could be used for screening and identification of modulators for either TRPV3 channel or other calcium permeable TRP channels.The identified natural neferine could be used as a tool for pharmacological investigations of TRPV3 channel function.