5-羟色胺1A受体激动剂高通量筛选模型的建立

目的为发现5-羟色胺1A(5-HT1A)受体的激动剂,通过报告基因活性检测的方法建立高通量筛选(HTS)细胞模型。方法将带有人源5-HT1A受体的真核表达质粒pcD-NA3.1(pcDNA3.1-h5-HT1AR)与带有报告基因pCRE-luc的pcDNA3.1质粒(pcDNA3.1-pCRE-luc)共转染到工具细胞中,通过对工具细胞选择、共转染质粒比例、化合物孵育时间及阳性化合物选择等条件进行探索和优化,建立了稳定表达人源5-HT1A受体并可用于该受体激动剂筛选的细胞模型(HEK293-h5-HT1AR)。结果①根据瞬转实验中信号值的高低,模型采用HEK293细胞作为工具细胞;②根据瞬转实验中的信噪比,发现pcDNA3.1-h5-HT1AR:pcDNA3.1-pCRE-luc的最佳比例为1∶3;③对化合物孵育时间进行优化,选择的最佳孵育时间为6 h;④阳性化合物的选择过程中,实验研究了5-HT.HCl,Flibanserin,8-OH-DPAT以及Lorcaser-in(APD356)4个已报道有5-HT1A受体激动活性的化合物,结果表明APD356在该体系中最适合作为阳性对照化合物。⑤该细胞株连续培养12代,信号稳定。结论通过对一系列实验条件进行选择和优化,建立了一个稳定表达人源5-HT1A受体的细胞模型,该模型可用于高通量5-HT1A激动剂的筛选。

Establishment of high throughput screening system of 5-HT1A receptor agonists

Aim To discover 5-HT 1A receptor agonists,a high throughput screening system of 5-HT 1A receptor was established by detecting the activity of reporter gene.Methods The pcDNA3.1-h5-HT 1A R plasmid and pcDNA3.1-pCRE-luc plasmid which harbored a luciferase reporter gene were co-transfected into appropriate cells,and then assay conditions were studied and optimized,such as the chosen of cell lines,the ratio of plasmids,incubation time and positive compounds.Results ① According to the signal efficacy,HEK 293 cells were chosen.② To get the best signal-to-noise ratio(S/N),the plasmid ratio between pcDNA3.1-h5-HT 1A R and pcDNA3.1-pCRE-luc was 1:3 during the transient transfection experiment.③ The best incubation time of compound was 6 hours.④ As to positive compounds,5-HT.HCl,Flibanserin,8-OH-DPAT and Lorcaserin(APD356) were studied respectively.The result showed that APD356 should be the best positive compound for this screening system.⑤ The cell line was cultured for twelve generations,and the signal was stable.Conclusion In this study,a cell line that stably expresses 5-HT 1A receptor is built,assay conditions are studied and optimized,and finally,it is proved that the cell line is suitable for high throughput screening of 5-HT 1A receptor agonists.