摘要
目的 以生长抑素受体2(SSTR2)为靶点,建立生长抑素类似物(SSTA)活性检测的细胞模型,为SSTR2激动剂以及SSTA的药物筛选提供一种简单稳定的评价方法。方法 将SSTR2目的基因整合入pEGFP-N3载体中,构建重组质粒SSTR2-pEGFP-N3,并转染进HEK293细胞,使用G418筛选后挑取阳性克隆,扩大培养获得稳转细胞株。通过荧光细胞成像、免疫蛋白印迹、qPCR等方法对获得的稳转细胞株进行鉴定。建立钙流检测体系,对该体系细胞数量、荧光指示剂浓度、孵育时间等条件进行优化。最后利用建立的筛选模型对不同批次的上市药物生长抑素制剂思他宁进行检测。结果 成功构建SSTR2稳转细胞株,受体主要分布在细胞膜上。确定钙流检测最优条件为:30 000个细胞每孔,Fluo-4/AM指示剂浓度为3 μmol·L -1 ~5 μmol·L -1 ,孵育时间为45 min。在该条件下,不同批次的思他宁EC 50 值稳定。结论 成功构建SSTR2稳转细胞株,并优化钙流检测方法,为生长抑素受体激动剂的筛选提供了一种简单稳定的模型。
Aim To establish a cell model to detect the activity of somatostatin (SST) by targeting somatostatin receptor 2 (SSTR2), and to provide a simple and stable evaluation method for the drug screening of SSTR2 agonists and somatostatin analogues (SSTA). Methods The target gene of SSTR2 was integrated into the pEGFP-N3 vector, and the recombinant plasmid was constructed and transfected into HEK293 cells. After G418 screening, positive clone was selected and the stable cell lines were obtained by expanding culture. The stable cell lines were identified by fluorescence cell imaging, Western blot and qPCR. A calcium flow detection system was established to optimize cell number, fluorescence dye concentration and incubation time. Finally, the screening model was used to detect the different batches of the marketed somatostatin preparation Stilamin. Results SSTR2 stable cell lines were successfully constructed, and the receptors were mainly distributed on the cell membrane. The optimal conditions for calcium flow detection were determined as follows: 30 000 cells/Well, Fluo-4/AM indicator concentration was 3 μmol·L^-1 ~5 μmol·L^-1 , incubation time was 45 min. Under this condition, EC 50 value of Stilamin in different batches was stable. Conclusions SSTR2 overexpressed stable cell lines are successfully constructed and calcium flow detection method is optimized to provide a simple and stable model for the screening of somatostatin receptor agonists.
作者
顾倩倩
刘翠
方伟彬
刘培庆
李民
GU Qian-qian;LIU Cui;FANG Wei-bin;LIU Pei-qing;LI Min(School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability andNew Drug Evaluation, Sun Yat-Sen University, Guangzhou 510006, China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2019年第11期1539-1544,共6页
Chinese Pharmacological Bulletin
基金
珠江人才计划本土创新科研团队项目(2017BT01Y093)
广东省自然科学基金资助项目(No 2016A030313335)