摘要
目的 建立神经介素NeuromedinU 2Receptor(NMU2R)激动剂的高通量筛选模型 ,从中药中筛选NMU2R受体激动剂。方法 将人NMU2R受体基因质粒 (hNMU2R/pCDNA3 1)与报告基因质粒 (MRE/CRE/LUC)按 1∶5的比例共转染到HEK2 93细胞 ,通过G4 18筛选 ,建立稳定的hNMU2R受体激动剂筛选细胞株。利用CRE的激活剂Forskolin和NMU2R内源激动剂NeuromedinU探索和优化荧光素酶表达时间、每孔细胞数目、荧光素酶底物浓度等筛选条件 ,建立可靠的筛选方法 ,并对部分中药水提液进行筛选。结果 已建立了一个稳定的hNMU2R受体激动剂筛选细胞株和可靠的筛选方法 ,其Z′ 因子为 0 75 ,荧光素酶表达时间为 6～ 8h ,细胞密度为每孔 4× 10 4～ 8× 10 4个。结论 该系统能够有效地应用于NMU2R受体激动剂的高通量筛选 ,能从中药中筛选出NMU2R受体的激动剂。
OBJECTIVE: To establish a high throughput screening method for finding agonist for NMU2R receptor. METHODS: The human NMU2R plasmid (hNMU2R/pCDNA3.1) and reporter gene plasmid (MRE/CRE/LUC) were cotransfected HEK293 to establish a cell line for agonist screening. To identify and optimize the assay condition, the effects of some factors on the assay were examined by using forskolin and neuromedin U, such as incubation time, cell number in each well and the concentration of luciferase's substrate. The extracts of some traditional Chinese medicine were screened. RESULTS: A steady cell line and a reliable method for NMU2R agonist screening were established. The Z'-factor value was 0.75. The agonist incubation time was 6 to 8 hours. The cell number per well was 4 × 104 to 8 × 104. CONCLUSION: This technology was successfully applied to identify agonist for NMU2R receptor from the traditional Chinese medicines.
出处
《中国药学杂志》
EI
CAS
CSCD
北大核心
2004年第3期185-188,共4页
Chinese Pharmaceutical Journal
基金
重庆市科委攻关项目 (渝科发计字 [2 0 0 2 ] 1号 )
重庆市计委重点高新技术项目 (渝计委计 [2 0 0 1] 1192号 )
