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目的:建立一种筛选评价雌激素相关受体α(estrogen-related receptorα,ERRα)调节剂的细胞模型,并用于计算机辅助设计、人工合成的化合物样品的评价。方法:用p CMV-h ERRα,3×ERRE-Luc及pRL-TK共同转染人胚肾细胞HEK-293,加入阳性药XCT790或筛选化合物孵育,分别检测HEK-293中萤火虫荧光素酶及海肾荧光素酶的活性,建立基于受体-应答元件的ERRα调节剂细胞筛选模型,用Z’因子评价本模型的稳定性,并测试了83个化合物对ERRα转录调节活性。用CCK-8检测DLE2-24对乳腺癌细胞MCF-7的增殖抑制效应,用RT-PCR考察DLE2-24对MCF-7中ERRα靶基因SOD2及VEGF的mRNA水平的影响。结果:成功建立ERRα-ERRE-Luc基因瞬时共转染模型,阳性药XCT790的IC50为0.24μmol·L-1,Z’因子为0.68,信号/本底为20.7。应用本模型筛选出15个ERRα反向调节剂。其中,DLE2-24剂量依赖性地抑制MCF-7的增殖,显著下调了ERRα下游靶基因SOD2及VEGF的mRNA水平。结论:本研究成功建立了一种高效、可靠的ERRα调节剂细胞筛选模型,适用于ERRα调节剂的发现与评价。
OBJECTIVE: To establish a cell model for screening and evaluating estrogen-related receptor α (ERRα) modulators, and to evaluate computer-aided design and synthetic compound samples. METHODS: Human embryonic kidney cell line HEK-293 was co-transfected with p CMV-h ERRα, 3 × ERRE-Luc and pRL-TK, and positive cells were treated with XCT790 or screened compound. Firefly luciferase Renal luciferase activity was assayed. A ERRα modulator cell screening model based on receptor-response element was established. The stability of this model was evaluated by Z ’factor. 83 compounds were tested for ERRα transcriptional activity. The inhibitory effect of DLE2-24 on the proliferation of breast cancer cells MCF-7 was detected by CCK-8, and the effect of DLE2-24 on the expression of ERRα target gene SOD2 and VEGF mRNA in MCF-7 was investigated by RT-PCR. Results: The co-transfection of ERRα-ERRE-Luc gene was successfully established. The IC50 of the positive drug XCT790 was 0.24μmol·L-1, the Z ’factor was 0.68 and the signal / background was 20.7. Fifteen ERRα reverse regulators were screened using this model. Among them, DLE2-24 dose-dependently inhibited the proliferation of MCF-7 and significantly down-regulated the mRNA levels of SOD2 and VEGF, a target gene of ERRα. Conclusion: This study successfully established an efficient and reliable cell model of ERRα regulator, which is suitable for the detection and evaluation of ERRα regulator.