Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay
Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay
复制标题
DOI：
10.1016/j.ejpb.2007.01.004
复制
发表时间：
2007-08-01
影响因子：
4.9
通讯作者：
Shi, Qunfang
中科院分区：
文献类型：
--
作者：
Li, Xinping;Shen, Fuwen;Shi, Qunfang
我们开发了一种基于功能细胞的高通量筛选（HTS）试验，以确定人类神经介肽亚型2受体的调节剂。该测定利用hNMU 2 R的信号转导途径，其正偶联腺苷酸环化酶和下游钙信号途径。我们详细描述了一个强大的，敏感的，和功能性的检测hNMU 2 R G蛋白偶联受体在人胚肾（HEK）-293细胞中表达，其活性反映了荧光素酶报告基因转录调控的3重复血清反应元件（SRE）-3重复多反应元件（MRE）-3重复环AMP（CAMP）反应元件（CRE）-VIP迷你启动子。用3 x SRE/3 x MRE/3 x CRE/VIP微型启动子驱动的荧光素酶和pCDNA3.1-NMU 2 R质粒稳定共转染的HEK 293克隆细胞系通过活性硫酸遗传霉素及其在已知激活细胞内信号转导的hNMU加毛喉素后用毛喉素攻击表达荧光素酶的能力进行选择。然后对筛选hNMU受体亚型2特异性激动剂的细胞密度、孵育时间、二甲基亚砜（DMSO）浓度进行优化，并考察中药提取物中的内源性发光物质是否干扰HEK 293细胞表达的荧光素酶的发光。发现最佳孵育时间为8 - 9 h，细胞密度和DMSO浓度分别优化为3 x 104 - 6 x 104和小于2%。我们的数据表明，hNMU 2 R luci-HEK 293细胞及其测定显示出低背景和高通量筛选的理想模型。这些结果表明，该报告基因测定可用于药理学分析，并且适用于人NMU 2 R激动剂的HTS。(c)2007 Elsevier B. V.保留所有权利。
We developed a functional cell-based high-throughput screening (HTS) assay to identify modulators of the human neuromedin subtype 2 receptor. This assay utilized the signal transduction pathway of hNMU2R, which is positively coupled to adenylyl cyclase and downstream calcium signal pathways. We describe in detail a robust, sensitive, and functional assay for the hNMU2R G-protein-coupled receptor expressed in human embryonic kidney (HEK)-293 cells, whose activity was reflected by a luciferase reporter gene transcriptionally regulated by a 3-repeat serum response element (SRE)-3 repeat multiple response element (MRE)-3 repeat cyclic AMP (CAMP) response element (CRE)-VIP mini promoter. The HEK 293 clonal cell line, stably co-transfected with the 3 x SRE/3 x MRE/3 x CRE/VIP mini promoter-driven luciferase and pCDNA3.1-NMU2R plasmid, was selected by active geneticin sulfate and their ability to express luciferase with a forskolin challenge following hNMU plus forskolin, known to activate intracellular signal transduction. Then the cell density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the hNMU receptor subtype 2 specific agonist were optimized, and whether intrinsic luminescent substance of extracts isolated from traditional Chinese herbs disturbs luminescence of luciferase expressed in HEK293 cells was considered. The optimal incubation time was found to be between 8 and 9 h, the cell density and DMSO concentrations were optimized from 3 x 104 to 6 x 104, and less than 2%, respectively. Our data show that hNMU2R luci-HEK293 cells and their assay exhibit a low background and ideal model for high-throughput screening. These results demonstrate that this reporter gene assay is useful for pharmacological analysis, and is amenable to HTS for human NMU2R agonists. (c) 2007 Elsevier B.V. All rights reserved.