基于报告基因的雌激素受体β亚型激动剂细胞筛选模型的建立  

Establishment of a reporter gene-based cell screening model for discovering new agonists of estrogen receptor β subtype

作　　者：陈立敏[1] 吕秋军[1] Satoshi Inoue 卞广兴[1] 陈振花[1] 温利青[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所,北京100850 [2]东京大学老年医学系

出　　处：《药学学报》2006年第8期721-726,共6页Acta Pharmaceutica Sinica

摘　　要：目的建立基于报告基因和雌激素受体β(estrogen receptorβ,ERβ)亚型信号通路的药物筛选模型,以期发现ERβ亚型激动剂。方法构建带有雌激素应答序列(estrogen responsive elem ent,ERE)靶序列和报告基因的诱导性表达载体pTAL-ERE-SEAP,并将其转入人胚肾(HEK293)细胞中,筛选报告基因分泌型碱性磷酸酶(secretedalkaline phosphatase,SEAP)表达受雌二醇(17β-estrad iol,E2)诱导的阳性克隆。选取相关核受体配体进行模型的特异性考察,同时考察模型的稳定性及时效量效关系,以及免疫细胞化学染色鉴定转染后ERβ的表达情况。利用此模型对本实验室样品库中的2 622个化合物进行筛选。结果转染pTAL-ERE-SEAP的HEK293细胞中,SEAP报告基因的表达受E2的诱导并呈剂量与时间依赖关系,E2对阳性克隆细胞无增殖作用,本模型的Z′因子值为0.7,免疫细胞化学染色结果表明,转染后ERβ表达、地塞米松以及其他配体的诱导表达率低。结论利用此模型通过测定SEAP报告基因的诱导表达水平可筛选ERβ亚型激动剂。Aim To establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor β subtype. Methods A recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector, pTAL-ERE-SEAP was then transfected into human embryonic kidney （HEK293） cells. C,418 （200 μg·mL^-1） was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERβ expression after transfection were observed. 2 622 compounds were screened by using this model. Results Stably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and timeeffect relationship manners. The Z＇ factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERβ. E2 had no proliferating effects on stably transfected clones. Conclusion Stably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor β subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.

关 键 词：雌激素 雌激素应答序列 分泌型碱性磷酸酶 药物筛选 报告基因 免疫细胞化学 

分 类 号：R965.1[医药卫生—药理学]