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目的 :建立靶向雌激素应答元件 (ERE)的药物筛选模型 ,为筛选雌激素受体 (ER)配体奠定基础。方法 :构建重组报告基因载体 pERE TAL SEAP ,与对照载体 pCMVβ瞬时共转染表达人ERα或ERβ的Hela细胞株 ,观察化合物对SEAP报告基因表达活性的影响。结果 :雌二醇 (E2 )诱导表达人ERα或ERβ的Hela细胞株SEAP的表达 ,EC50 分别为 (80 .5 8± 8.5 1) pmol·L-1和 (10 3.90± 5 .2 9) pmol·L-1,最大效应浓度为 10nmol·L-1;金雀黄素 (Gen)也诱导表达人ERα和ERβHela细胞株SEAP的表达 ,EC50 分别为 (39.38± 2 .2 6 )nmol·L-1和 (10 .86±0 .75 )nmol·L-1;最大效应浓度为 1μmol·L-1;E2 和Gen诱导SEAP表达的最大水平较对照孔细胞高 7～ 14倍。ER拮抗剂ICI182 ,780可完全抑制E2 和Gen对SEAP的诱导作用。结论 :利用此模型检测化合物诱导报告基因的表达水平可筛选和发现ER配体。
Objective: To establish a drug screening model targeting estrogen response element (ERE) and lay the foundation for the screening of estrogen receptor (ER) ligand. Methods: The recombinant reporter gene vector pERE TAL SEAP was constructed and transiently transfected into Hela cells expressing human ERα or ERβ with control vector pCMVβ. The effects of compounds on the expression of SEAP reporter gene were observed. Results: Estradiol (E2) induced the expression of SEAP in Hela cells expressing human ERα or ERβ with EC50 of (80.58 ± 8.5 1) pmol·L -1 and (10 3.90 ± 5.92) pmol, respectively · L-1, the maximum concentration of 10nmol·L-1; geniposide also induced the expression of SEAP in human ERα and ERβHela cell lines with EC50 of (39.38 ± 2.26) nmol·L- 1 and (10.86 ± 0.75) nmol·L-1, respectively, and the maximum concentration of 1 μmol·L-1. The maximum level of SEAP expression induced by E2 and Gen was 7-14 fold higher than that of control cells. ER antagonist ICI182,780 completely inhibited the induction of SEAP by E2 and Gen. CONCLUSIONS: The use of this model to detect compound-induced reporter gene expression levels can screen and discover ER ligands.