摘要
目的建立不同分子作用机制的糖皮质激素受体α（GRα）调节剂高通量筛选模型，筛选新型糖皮质激素受体调节剂。方法克隆 GRα的配体结合域 LBD 和全长基因，构建哺乳动物细胞表达载体 pBIND-GAL4-GRα LBD、pTARGET-GRα，分别与已构建的含 GAL4响应元件5× UAS 的荧光素酶报告质粒 p5× UAS-luc 和含有4× GRE 的报告质粒pGRE-luc 共转染 HeLa 细胞，建立两种不同机制的报告基因细胞筛选方法，通过报告基因的表达检测化合物对 GRα受体转录调控功能的调节剂作用；利用实时定量 PCR 方法进一步验证化合物对糖皮质激素受体靶基因 mRNA 水平的调控作用。 结果经过分别共转染表达质粒和报告质粒，GRα激动剂地塞米松可剂量依赖地诱导5× UAS-luc 和4× GRE-luc两个模型的荧光素酶的表达，在5× UAS-luc 模型中，最大上调倍数可达（9.0±0.2）倍，EC50值为（0.28±0.07）mmol/L， Z＇因子为0.52。在4× GRE-luc 模型中，最大上调倍数可达（3.2±0.3）倍，EC50值为（1.81±0.13）mmol/L，Z＇因子为0.49。利用模型 pBIND-GAL4-GRαLBD/p5× UAS-luc从2000多个微生物和植物来源的天然以及合成化合物中筛选得到1个微生物来源的天然产物黑麦酮酸 D，黑麦酮酸 D 对 GRα具有2-3倍的激动活性。进一步的定量PCR 的结果说明黑麦酮酸 D 能有效上调多个 GRα调控的靶基因的表达。 结论两种报告基因筛选方法灵敏、稳定，可以用于 GRα调节剂的高通量筛选。
Objective To develop high throughput screening models for glucocorticoid receptor α （GRα） modulators and discover novel modulators of GRα.
Methods The ligand binding domain （LBD） fragment and full-length gene of GRαwere amplified by real time-PCR from human hippocampal total mRNA to construct chimera expression plasmid pBIND-GAL4-GRα LBD and full length express plasmid pTARGET-GRα. The screening models were developed by transfecting the HeLa cells, and the efficiency of modulators on the transactivities of GRα was assayed by measuring the reporter luciferase gene expression. The mRNA levels of targeting genes of GRαin cells were analyzed by real time-PCR.
Results Dexamethasone, a known GRαagonist, could induce the expression of luciferase in a dose-dependent manner in the both screening models. For the 5 ×UAS-luc model, the maximum fold of induction was about （9.0± 0.2）, the EC50 value was about （0.28 ± 0.07） mmol/L, and the parameter Z＇-factor value was about 0.52. In the 4 × GRE-luc model, the maximum fold of induction was about （3.2 ± 0.3）, the EC50 was （1.81 ± 0.13） mmol/L, and the parameter Z＇-factor value was 0.49. After screening, one microbial metabolite secalonic acid D from 2000 compounds was identified as a GRα agonist, it could significantly induce the luciferase expression, and the maximum induction folds was about 2 - 3 on both assays. The further real time-PCR results showed that secalonic acid D could increase the mRNA levels of GRαtarget genes F2R, NPY2R, ULBP3 and OR8D4 in the treated cell line LO2. Conclusion The two screening models are robust and sensitive, and can be used to discover the GRα new modulator. Secalonic acid D has been discovered as a new GRαagonist.
出处
《中国医药生物技术》
2014年第2期110-115,共6页
Chinese Medicinal Biotechnology
基金
国家自然科学基金(81173137)
"重大新药创制"国家科技重大专项(2012ZX09301002-003)
河北省科技支撑计划重点项目(13272607D)
关键词
糖皮质激素受体Α
生物应答调节剂
降血糖药
高通量筛选
Glucocorticoid receptor α
Biological response modifiers
Hypoglycemic agents
High throughout screening
