基于HTRF方法PGE2释放抑制高通量筛选模型建立及其在中药活性成分发现中应用.docVIP

基于HTRF方法PGE2释放抑制高通量筛选模型建立及其在中药活性成分发现中应用 　　[摘要]前列腺素E2（ prostaglandins E2， PGE2）是参与炎症、疼痛等多种生理病理机制过程的活性物质。该文在脂多糖（lipopolysaccharide，LPS）诱导小鼠巨噬细胞RAW264.7 产生PGE2的模型上， 利用均相时间分辨荧光（homogeneous time-resolved fluorescence，HTRF）方法结合全自动化筛选操作系统，建立检测PGE2含量的高通量模型，选用公司中药物质基础库内的5 121个中药成分为检测样品，用于筛选PGE2抑制剂。该模型选择细胞铺板密度为每孔2 000个细胞； 阳性化合物的IC50平均值为（7.3±0.1） μmol；检测板的Z′因子均大于0.5，平均值为0.7；5 121个样品中有228个样品在检测浓度对PEG2产生的抑制率大于50%，23个样品对PEG2产生的抑制作用大于80%。该模型数据稳定性和准确性均达到预期标准，表明筛选结果可靠真实；自动化筛选系统的引入，令该模型具有快速，微量，高效等优势，日平均筛选量达到14 000数据点以上，为抗炎镇痛相关新药发现和中药有效物质成分的前期快速筛选提供了新的模型。 　　[关键词]前列腺素E2；HTRF；高通量；中药物质基础库 　　[Abstract]Prostaglandin （PG） E2 is an active substance in pathological and physiological mechanisms， such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence（HTRF） PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps， including the cell density optimization and IC50 values determination of a positive compound， were taken to verify the stability and sensibility of the assay. Low intra-plate， inter-plate and day-to-day variability were observed in this 384-well， high-throughput format assay. Totally 5 121 samples were selected from the company′s traditional Chinese medicine（TCM） material base library and used to screen PGE2 inhibitors. In this model， the cell plating density was 2 000 cells for each well； the average IC50 value for positive compounds was （7.3±0.1） μmol； the Z′ factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples， 228 components exhibited a PGE2 production prohibition rate of more than 50%， and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy， indicating the reliability and authenticity of the scre