组胺H3受体激活剂高通量筛选细胞模型的研究

摘 要 目的 建立基于报告基因的组胺H3受体（H3R）激活剂的高通量筛选模型，用此模型对收集到的中草药化合物组分进行筛选，以发现新的组胺H3R激活剂。 方法 将H3R基因质粒（H3R/pCDNA3.1-hygro）与报告基因质粒（3XCRE-LUC）按3：1的比例共转染入HEK293细胞，建立了稳定的H3R配体的报告基因筛选细胞株。激活剂与细胞表面H3R结合后，激活相应的信号通路，调节Forskolin刺激后的报告基因的表达，通过测定荧光素酶报告基因表达水平的变化，评估激活剂影响H3受体的生物活性。 结果 通过对筛选条件，如激活剂孵育时间、Forskolin终浓度、化合物溶剂的选择、溶剂DMSO终浓度等的优化，建立了可靠的筛选方法，并对多种中草药萃取物进行了筛选，找到了两种对H3R有活性的中药组分。结论 建立的细胞模型可以有效的应用于以组胺H3受体为靶点的高通量药物筛选。

Establishment of a Cell Based High Throughput Screening Model for Discovering New Agonists of Histamine H3 Receptor

ABSTRACT: AIM A cell based reporter gene assay had been established and optimized for high throughput screening to find agonists for human histamine 3 receptor (H3R) from Chinese herb components. METHODS The H3 receptor plasmid (H3R/pCDNA3.1-hygro) and reporter gene plasmid (3XCRE-LUC) were cotransfected HEK293 and a stable cell line was selected with antibiotics for H3 receptor agonist screening. Upon agonist binding, H3 receptor was activated and lead to a decrease of the high expression of luciferase gene induced by forskolin. RESULT To identify and optimize the assay condition, the effects of some factors were examined, such as compound solvent selection, final concentration of forskolin, agonist and forskolin incubation time, final concentration of DMSO. A steady cell line and a reliable method were established for H3R agonist screening, the Z’factor value was above 0.5, the signal did not change using HEK/H3R/CRE cell line passage 5 to passage 20. By using this assay system, two herb components were identified and regarded to contain agonists for H3 receptor. CONCLUSION This drug screening cell model has been successfully used for histamine 3 receptor target high throughput drug screening.