论文部分内容阅读
多巴胺D5受体是属于多巴胺能D1样受体的一种亚型,在多种神经退行性疾病中具有重要功能。天然产物中的一些活性成分可能是该受体的激动剂,可应用于相关疾病的治疗。为了从天然产物库筛选能激活D5受体的天然活性成分,将人多巴胺受体D5R基因质粒(hD5R/pcDNA3.1)与报告基因质粒(4×CRE/TK/Luci/pGL3)共转染HEK293细胞,通过G418筛选后,挑取单克隆细胞,并用CRE(cAMP response element)响应阳性药forskolin和D5受体特异性激动剂SKF38393进行刺激,建立D5受体激动剂高通量筛选模型的最佳条件。结果显示:实验获得了多株D5受体单克隆细胞。其中,编号为9-22的D5受体单克隆细胞株最佳筛选条件是:细胞接种浓度为5×104/ml,forskolin有效刺激浓度为5～20μmol/L,D5受体激动剂SKF38393有效刺激浓度为100nmol/L～100μmol/L。利用该细胞筛选模型对200余种天然产物水提物进行了初步筛选,结果筛选出了3种天然产物水提物具有明显的激活效应,有可能包含有D5受体的激动剂,为D5受体激动剂的筛选打下基础。
Dopamine D5 receptors belong to a subtype of dopaminergic D1-like receptors and have important functions in a variety of neurodegenerative diseases. Some of the active ingredients in natural products may be agonists of this receptor and may be used in the treatment of related diseases. To screen natural product libraries for natural active ingredients that activate D5 receptors, the human dopamine receptor D5R gene plasmid (hD5R / pcDNA3.1) was co-transfected with the reporter plasmid (4 × CRE / TK / Luci / pGL3) into HEK293 After selection by G418, the monoclonal cells were picked and stimulated with CRE (cAMP response element) -responsive forskolin and D5 receptor-specific agonist SKF38393 to establish the best high-throughput D5 receptor agonist screening model condition. The results showed that multiple D5 receptor monoclonal cells were obtained. Among them, the optimal screening conditions for D5 receptor monoclonal cell line numbered 9-22 were: cell seeding concentration 5 × 104 / ml, forskolin effective stimulus concentration 5 ~ 20μmol / L, D5 receptor agonist SKF38393 effective stimulus The concentration of 100nmol / L ~ 100μmol / L. The cell screening model was used to screen more than 200 natural product water extracts. As a result, three natural product water extracts were screened for obvious activation effects, possibly including D5 receptor agonists, The screening of body agonists lays the foundation.