多巴胺(D5)受体激动剂高通量筛选模型的建立和天然活性成分的筛选

High Throughput Screening Method of Identifying D5 Receptor Agonist from Chinese Herbs

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。

D5 receptor is a subtype of dopamine D1-like receptor,and it plays a functional role in many neurological disorders. Some natural compounds from Chinese herbs,which were shown to have the property as that of receptor agonist,might provide a rich source in search of new candidates for therapeutic use. For exploring this possibility,we developed a cell-based high throughput （HTS） D5 receptor assay to screen the herb-based natural compound library established in our centre. The D5 receptor plasmid （hD5R/ pcDNA3.1） and reporter gene plasmid （4×CRE/TK/Luci/pGL3） were co-transfected into HEK293 cell line. After G418 being selected,the monoclonal cell lines bearing hD5R and the reporter gene were established and used for agonist screening. To optimize the assay condition,the effects of some factors such as cell number per well,incubation time,and the doses of SKF38393 （a potent selective partial D1-like agonist） were examined by using forskolin,a positive compound for cAMP response element. The best condition for this HTS assay included：the cell number at 5×104 /mL,the dose of forskolin at 5-20 μmol/L,the dose of SKF38393 at 100 nmol/L-100 μmol/L,and the agonist incubation time at 6-8 h. Thereafter,water extracts from more than 200 Chinese herbs in our library were screened and three of these water extracts showed positive activity,with higher or similar activity as SKF38393. In conclusion,we have established a cell-based HTS assay for D5 receptor agonist screening,and by use of this HTS assay,3 Chinese herbs maybe contain components exhibiting D5 receptor agonist property. This work provides an alternative vision of how to use herb medicines and a way to develop potential drugs for treatment of neurological disorders.