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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IC_(50)为24.5±4.1μM(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TRPV1和TRPV4通道无抑制作用。总之,实验数据显示,Flex Station3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。
In order to establish a high-throughput screening method for TRPV3 channel modulators, we optimized the conditions of HEK293T cells transiently transfected with TRPV3 channels, Cal-520 dye concentration and dye incubation time using FlexStation3 microplate reader. The results showed that 40 000 cells / well, 5.0% Cal-520 dye concentration and 1.5 h dye incubation time were the best experimental conditions. The screening method was used to screen and discover that neferine, an active ingredient from Nymphaeaceae, can inhibit TRPV3 channel. The inhibition was confirmed by whole-cell patch clamp assay and its IC50 was 24.5 ± 4.1 μM (n = 6). Selective electrophysiological evaluation of other TRPV channel members showed that neferine did not inhibit TRPV1 and TRPV4 channels. In conclusion, experimental data show that Flex Station 3 high-throughput calcium fluorescence screening can be used to find TRPV3 and other TRP channel modulators. The purified natural product neferine screened can be used as a drug tool for the TRPV3 channel to study the pharmacological function of channels.