论文部分内容阅读
目的:建立一种以共转染为基础的、高效灵敏的植物雌激素活性成分的细胞筛选方法,应用此方法研究鹰嘴豆提取部位的雌激素效应。方法:RT-PCR方法扩增人雌激素受体α(hERα)cDNA,并构建哺乳细胞表达载体pERα。将此载体与含有雌激素应答序列(3×ERE)的Luc报告基因载体(pERE-Luc)以不同比例共转染MCF-7细胞,比较不同比例下Luc的活力,确定最佳共转染比例。用芒柄花素、鹰嘴豆豆芽素A和染料木素等植物雌激素验证了该模型的灵敏性,并进一步测定了鹰嘴豆不同提取部位的Luc活力。结果:将pERα与pERE-Luc共转染MCF-7细胞,与pERE-Luc单转染相比,显著提高Luc的活力,且在10∶1(pERE-Luc∶pERα)时活力最高,Luc活力提高了5倍。用此转染比例测定芒柄花素、鹰嘴豆豆芽素A和染料木素等植物雌激素Luc活力测定结果表明,共转染能诱导Luc的表达,且ER特异性抑制剂ICI 182,780能抑制其活性。利用此模型发现鹰嘴豆的70%乙醇总提取物、乙酸乙酯部位和石油醚部位均具有大量的雌激素活性物质。ICI 182,780能有效抑制其雌激素效应。结论:成功建立了一种共转染为基础的植物雌激素活性成分的筛选方法,该方法具有较高的特异性和灵敏性,可用于植物雌激素活性成分的筛选。
OBJECTIVE: To establish an efficient and sensitive cell-based screening method for phytoestrogen active ingredients based on co-transfection, and to study the estrogen effect of chickpea extract using this method. Methods: Human estrogen receptor α (hERα) cDNA was amplified by RT-PCR and the mammalian expression vector pERα was constructed. The vector was co-transfected into MCF-7 cells with Luciferase reporter assay (pERE-Luc) containing estrogen response sequence (3 × ERE), and the luciferase activities of different proportions were compared to determine the optimal cotransfection ratio . The sensitivity of this model was validated by phytoestrogens such as mangosteen, chickpea bean buds A, and genistein, and the Luc activity in different extracts of chickpea was further determined. Results: The co-transfection of pERα and pERE-Luc into MCF-7 cells significantly increased the activity of Luc compared with that of pERE-Luc, and the highest activity was found at 10: 1 (pERE-Luc: pERα) Increased by 5 times. Using this transfection ratio, we determined the Luc activities of phytoestrogens such as formononetin, chickpea bean budsidin A and genistein, and found that co-transfection could induce the expression of Luc, and the ICR 182,780, an inhibitor of ER, could inhibit Its activity. Using this model, we found that 70% ethanolic total extract of chickpea, ethyl acetate fraction and petroleum ether fraction have a large amount of estrogen active substances. ICI 182,780 can effectively inhibit its estrogen effect. Conclusion: A cotransfection-based screening method for phytoestrogen active ingredients has been successfully established. The method has high specificity and sensitivity and can be used for the screening of phytoestrogen active ingredients.