以中链脂肪酸受体GPR84为靶点的药物高通量筛选模型的建立和应用

Establishment and Application of a High-throughput-screening Model for Medium-chain Fatty Acid-sensing Receptor GPR84

G蛋白偶联受体84(GPR84)是C9-C14的中链脂肪酸受体,不仅在脂肪酸代谢和机体免疫反应中发挥着重要作用,而且与多发性硬化症、内毒素血症等炎症性免疫疾病有着密切联系.因此,找到以GPR84为靶点的配体,可能为治疗这些疾病提供一种有效的方法.构建稳定过表达GPR84受体蛋白的细胞系,为筛选GPR84为靶点的配体和研究这些免疫疾病提供了重要途径.本文将含GPR84和Gα16基因质粒共转染到HEK293细胞中,经过抗生素抗性筛选,挑取稳定表达GPR84蛋白的HEK293细胞系.用RT-PCR、免疫荧光染色等实验方法检测了GPR84基因和其编码受体蛋白的表达;用钙流实验、环腺苷酸实验、蛋白质印迹分析、流式细胞分析证实了表达的GPR84具有完整的生物学活性,并利用该细胞系进行了药物高通量筛选,得到了新的GPR84拮抗剂,为进一步阐明GPR84的生物学功能,研究并治疗与其相关免疫疾病奠定了基础.

G protein coupled receptor 84（GPR84） is medium-chain fatty acid-sensing receptor（C9~C14）. It plays an important role in metabolism of fatty acid and immune response, and it＇s also deeply involved in inflammatory immune diseases such as multiple sclerosis and endotoxemia. It may provide an effective method to treat these diseases by finding GPR84 ligands. It＇s important to construct a cell line stably expressing human GPR84 for screening its ligands and researching diseases. In this research, we transfected plasmids encoding GPR84 and Gtx 16 proteins into HEK293 cells. After selection with antibiotics, we picked out monoclones which stably expresses GPR84 to form cell lines. The permanent expression of exogenous GPR84 proteins in HEK293 cells was detected by RT-PCR, immunofluorescence staining, calcium mobilization and cAMP assay. Western blot and FACS were conducted to examine the activity of GPR84. Above all the results demonstrate that GPR84 receptor has excellent biology activity. Based on cell lines stably expressing GPR84, we conducted high-throughput assay for the screening of GPR84 ligands and found new GPR84 antagonists. We can further research on the biological functions of GPR84 and immune diseases targeted GPR84 with these antagonists.