毒蕈胆碱M_1受体激动剂的高通量筛选模型

目的　建立毒蕈碱样胆碱M1 受体激动剂的高通量筛选模型 ,以此模型进行M1 受体激动剂筛选。方法　将M1 受体基因质粒 (M1 /pCDNA3 1 )与报告基因质粒 (3×CRE/ 3×MRE/SRE LUC)按 1∶5的比例共转染HEK2 93 ,建立了一个稳定的M1 受体激动剂报告基因筛选细胞株。配体与细胞表面M1 受体结合后 ,激活相应的信号通路 ,调节报告基因的表达 ,通过测定荧光酶素报告基因表达水平的变化 ,评估配体激活M1 受体的生物活性。结果　通过对筛选条件 ,如细胞数目、荧光素酶表达时间、底物浓度的优化 ,建立了可靠的筛选方法 ,并对多种抗衰老中药水提物进行了筛选 ,找到 3种对M1 受体有活性的中药。结论　该系统能准确、稳定、有效地应用于M1 受体激动剂的高通量筛选

High throughput screening method of identifying agonist for muscarinic receptor

AIM A reporter gene assay has been established and optimized for high throughput screening to identify human muscarinic 1 receptor (M 1). METHODS The M 1 receptor plasmid (M 1/pCDNA3 1) and reporter gene plasmid (3×CRE/3×MRE/SRE LUC ) were cotransfected into HEK293 to establish a stable cell line for M 1 receptor agonist screening. Upon ligand binding, M 1 receptor was activated and lead to the activation of luciferase gene. RESULTS To identify and optimize the assay condition, some factors, such as cell number in each well, substrate concentration and incubation time, were examined and optimized. Four hundred extracts of traditional Chinese medicine were screened. By using this technology , three extracts of TCM were identified to containe agonists for M 1 receptor. CONCLUSION The reporter gene assay technology has been successfully applied to identify agonist for M 1 receptor.