作者感谢戴尔·莱特曼在使用瞬态透切检测来确定灵长类植物食物的雌激素活性方面进行了初步培训。感谢布拉德福德·韦斯特里奇和C·埃里克·约翰逊帮助建立实验室设备，并培训学生提取方法。最后，感谢印第安纳大学为这项研究提供资金。

1. 植物材料的准备
<ol><li>冷冻使用嗜酸剂新鲜收集的干燥植物物品。<ol>
<li>为了保护样品免受光线照射，在干燥过程中用铝箔盖住腔室。</li>
		<li>为了确保样品完全干燥，嗜酸，直到室不再感到寒冷触摸和植物材料不再失去质量时称重。</li>
		<li>将干燥的植物存放在无菌低残留袋中，在没有光线的情况下，直到研磨。</li>
	</ol>
</li>
	<li>使用带 0.85 mm 网格屏幕的研磨机精细研磨样品。<ol>
<li>在没有光线的情况下将地面样品存放在袋子里，直到提取。</li>
	</ol>
</li>
</ol>2. 提取植物二次代谢物
<ol><li>要提取二级植物代谢物，请使用 1 克干样品与 10 mL 的 HPLC 级甲醇的比例。<ol>
<li>在分析平衡上称量样品，并将其添加到大小合适的埃伦迈尔烧瓶（125-250 mL）中。然后添加适量的甲醇。记录提取的样本的质量。</li>
		<li>用铝箔盖住植物甲醇溶液，然后在室温 （RT） 下以 100 rpm 的速度在轨道摇床上旋转 3 天，使潜在的雌激素化合物溶解到甲醇中。</li>
		<li>使用滤纸（125毫米）将超细长剂倒入滴滤系统。</li>
		<li>使用旋转蒸发器，将植物提取物干燥，直到样品变厚，但可倒入 300 mL 的圆底烧瓶中。将样品倒入 50 mL 圆底烧瓶中，用少量甲醇冲洗大烧瓶。继续在小烧瓶中干燥样品，直到甲醇完全蒸发。</li>
		<li>使用分析平衡对样品残留物进行称重。记录残留质量。</li>
		<li>将植物提取物溶解在二甲基硫化物 （DMSO） 中，浓度为 0.1 克提取物到 2 mL DMSO。漩涡，直到同质化。</li>
		<li>将植物提取物-DMSO溶液在4°C下存放在琥珀色玻璃瓶中，直到检测。</li>
	</ol>
</li>
</ol>警告:植物可以产生未知的生物活性化学物质，DMSO是一种可以通过细胞膜运输它们的工具。在处理这些样品时，使用适当的个人防护设备并小心谨慎。
3. 人体雌激素受体β转位检测12
注:检测协议的第 1 天需要无菌技术和层流罩。
<ol><li>为标准曲线准备 17+ 雌二醇的稀释。<ol>
<li>将细胞恢复介质和复合筛选介质 （CSM） 从冰柜存储中转移，并在 37 °C 水浴中解冻。</li>
		<li>标记微中心管中间体 1 和 2 （INT1，INT2） 和 1-8。</li>
		<li>用 995 μL 的 CSM 填充 INT1，用 615 μL 的 CSM 填充 INT2，用 900 μL 的 CSM 填充 1 管，用 600 μL 的 CSM 填充 2-8。将管 8 放在一边。</li>
		<li>将 100 μM 17+Estradiol 库存的 5 μL 转入 INT1。丢弃提示。涡。</li>
		<li>每次传输前，冲洗移液器 3 次，然后从 INT1 传输 10 μL 到 INT2。丢弃提示。</li>
		<li>冲洗移液器3次，然后从INT2传输100μL到管1。丢弃提示。将 300μL 从 1 管转移到 2 管中。重复管 3 到 7。将 300μL 从 7 管中丢弃到废物容器中。管8是零，不接收雌二醇。镀膜标准的最终浓度为:400、133.3、44.44、14.815、4.938、1.646、0.5487和0 pM雌二醇。</li>
	</ol>
</li>
	<li>准备样品化合物。<ol>
<li>漩涡样本。</li>
		<li>以 DMSO 中每个植物样本的 4 μL 为例，并添加到 496 μL 的 CSM 中，以产生 0.8% 的 DMSO 解决方案。</li>
	</ol>
</li>
	<li>迅速解冻记者细胞。<ol>
<li>从 37 °C 水浴中取出细胞恢复介质管。使用 70% 乙醇对外部表面进行消毒。</li>
		<li>通过将预加热的 CRM 的 10 mL 传输到冷冻细胞管中，从 -80 °C 存储中检索记者细胞并解冻。</li>
		<li>关闭记者细胞管，转移到 37°C 的水浴室 5-10 分钟。</li>
		<li>从水浴中取出记者细胞悬浮管。轻轻倒置细胞管数次，以分解细胞聚集物并产生同质悬浮物。用 70% 乙醇清洁管子表面。</li>
	</ol>
</li>
	<li>检测电镀<ol>
<li>使用多通道移液器将 100 μL 的记者细胞悬浮液分配到每个井中。</li>
		<li>将 100 μL 的样品分三次放入适当的检测井中。</li>
		<li>将板转移到 37 °C 中，加湿 5% CO2 孵化器 22-24 小时。</li>
	</ol>
</li>
	<li>在黑暗的冰箱中通宵解冻检测基板和检测缓冲区，为第 2 天做准备。</li>
	<li>在板式孵化结束之前，从冰箱中取出检测基板和检测缓冲区，放置在低光区域，直到平衡到 RT。一旦在RT，轻轻倒置每个管几次，以彻底混合解决方案。<ol>
<li>在孵化完成之前，将检测缓冲区的全部内容倒入检测基板管中，以创建 Luciferase 检测试剂。轻轻混合，以免产生泡沫。</li>
		<li>孵化完成后，倒置板将内容倒置到适当的废容器中。轻轻敲击干净的吸水纸巾上的板，从井中取出最后一滴水滴。</li>
		<li>在每个油井中添加 100μL 的卢西费酶检测试剂。让检测板在RT上休息15分钟。不要摇盘子。</li>
	</ol>
</li>
	<li>使用 96 井板读数氧化铝计量化发光。</li>
</ol>

<ol>
	<li>Wasserman, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+plant+consumption+predicts+red+colobus+monkey+(Procolobus+rufomitratus)+hormonal+state+and+behavior.">Estrogenic plant consumption predicts red colobus monkey (Procolobus rufomitratus) hormonal state and behavior.</a> Hormones and Behavior. 62 (5), 553-562 (2012).</li><li>Wasserman, M. D., Milton, K., Chapman, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+phytoestrogens+in+primate+ecology+and+evolution.">The roles of phytoestrogens in primate ecology and evolution.</a> International Journal of Primatology. 34 (5), 861-878 (2013).</li><li>DeGabriel, J. L., Moore, B. D., Foley, W. J., Johnson, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+plant+defensive+chemistry+on+nutrient+availability+predict+reproductive+success+in+a+mammal.">The effects of plant defensive chemistry on nutrient availability predict reproductive success in a mammal.</a> Ecology. 90 (3), 711-719 (2009).</li><li>Wasserman, M. D., Steiniche, T., Despr&#233;s-Einspenner, M. -. L., Lambert, J. E., Rothman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Diet &amp; Nutrition. , (2020).</li><li>Benavidez, K. M., Chapman, C. A., Leitman, D. C., Harris, T. R., Wasserman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intergroup+variation+in+oestrogenic+plant+consumption+by+black-and-white+colobus+monkeys.">Intergroup variation in oestrogenic plant consumption by black-and-white colobus monkeys.</a> African Journal of Ecology. , (2019).</li><li>Bennetts, H. W., Underwood, E. J., Shier, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+breeding+problem+of+sheep+on+subterranean+clover+pastures+in+Western+Australia.">A specific breeding problem of sheep on subterranean clover pastures in Western Australia.</a> Australian Veterinary Journal. 22 (1), 2-12 (1946).</li><li>Tubbs, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenicity+of+captive+southern+white+rhinoceros+diets+and+their+association+with+fertility.">Estrogenicity of captive southern white rhinoceros diets and their association with fertility.</a> General and Comparative Endocrinology. 238, 32-38 (2016).</li><li>Shen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+the+influences+of+diosgenin+on+aging+ovarian+reserve+and+function+in+a+mouse+model.">Observation of the influences of diosgenin on aging ovarian reserve and function in a mouse model.</a> European Journal of Medical Research. 22 (1), 42 (2017).</li><li>Bou&#233;, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+estrogenic+effects+of+legume+extracts+containing+phytoestrogens.">Evaluation of the estrogenic effects of legume extracts containing phytoestrogens.</a> Journal of Agricultural and Food Chemistry. 51 (8), 2193-2199 (2003).</li><li>Klinge, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+receptor+interaction+with+estrogen+response+elements.">Estrogen receptor interaction with estrogen response elements.</a> Nucleic Acids Research. 29 (14), 2905-2919 (2001).</li><li>Nishikawa, J. -. i., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+screening+methods+for+chemicals+with+hormonal+activities+using+interaction+of+nuclear+hormone+receptor+with+coactivator.">New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator.</a> Toxicology and Applied Pharmacology. 154 (1), 76-83 (1999).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Estrogen Receptor Beta (ERb; ESR2; NR3A2) Reporter Assay System. , (2020).</li><li>Wasserman, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+plant+foods+of+red+colobus+monkeys+and+mountain+gorillas+in+uganda.">Estrogenic plant foods of red colobus monkeys and mountain gorillas in uganda.</a> American Journal of Physical Anthropology. 148 (1), 88-97 (2012).</li><li>Vivar, O. I., Saunier, E. F., Leitman, D. C., Firestone, G. L., Bjeldanes, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+activation+of+estrogen+receptor-&#946;+target+genes+by+3,+3'-diindolylmethane.">Selective activation of estrogen receptor-&#946; target genes by 3, 3'-diindolylmethane.</a> Endocrinology. 151 (4), 1662-1667 (2010).</li><li>Whitten, P. L., Patisaul, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+and+interassay+comparisons+of+phytoestrogen+action.">Cross-species and interassay comparisons of phytoestrogen action.</a> Environmental Health Perspectives. 109, 5-20 (2001).</li><li>Di Gioia, F., Petropoulos, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Food and Nutrition Research. , (2019).</li><li>Lutz, I., Kloas, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphibians+as+a+model+to+study+endocrine+disruptors:+I.+Environmental+pollution+and+estrogen+receptor+binding.">Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding.</a> Science of The Total Environment. 225 (1), 49-57 (1999).</li><li>Felcyn, J. R., Davis, J. C. C., Tran, L. H., Berude, J. C., Latch, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aquatic+Photochemistry+of+Isoflavone+Phytoestrogens:+Degradation+Kinetics+and+Pathways.">Aquatic Photochemistry of Isoflavone Phytoestrogens: Degradation Kinetics and Pathways.</a> Environmental Science &amp; Technology. 46 (12), 6698-6704 (2012).</li><li>Jeng, Y. -. J., Kochukov, M. Y., Watson, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+estrogen+receptor-alpha-mediated+nongenomic+actions+of+phytoestrogens+in+GH3/B6/F10+pituitary+tumor+cells.">Membrane estrogen receptor-alpha-mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells.</a> Journal of Molecular Signaling. 4, 2-2 (2009).</li><li>Dixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytoestrogens.">Phytoestrogens.</a> Annual Review of Plant Biology. 55, (2004).</li><li>Kuiper, G. G. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+Estrogenic+Chemicals+and+Phytoestrogens+with+Estrogen+Receptor+&#946;.">Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor &#946;.</a> Endocrinology. 139 (10), 4252-4263 (1998).</li><li>Wasserman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Feeding on Phytoestrogens: Implications of Estrogenic Plants for Primate Ecology. , (2011).</li><li>Jefferson, W. N., Patisaul, H. B., Williams, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+consequences+of+developmental+phytoestrogen+exposure.">Reproductive consequences of developmental phytoestrogen exposure.</a> Reproduction. 143 (3), 247-260 (2012).</li></ol>

作者没有什么可透露的。

ER+记者检测开发为单独筛选药物制剂也适用于筛选植物性植物雌激素通过 ER+生物活性的食物。协议中的重要考虑因素包括小心处理植物样品:需要迅速干燥新鲜植物材料以防止成型或其他生物退化，并且需要远离光线以防止化合物18的光解。制造商提供的检测协议12 是明确的，需要很少的修改进行筛选。制造商建议的标准曲线已在此协议中进行了修改，以增加曲...

植物是许多动物必需的食物来源，提供对生存、繁殖、生长、发育和行为至关重要的卡路里和营养物质。植物产生成千上万的化学物质，许多是适应自身生长、植物维持和繁殖的。其他化合物，被视为植物二次代谢物（PSM），其功能不太清晰，虽然有些是有毒的，并可能用作防御食草动物和寄生虫（如生物碱，单宁）2，3.其中一些化学物质有能力影响动物的长期生理过程，如内分泌功能，虽然为什么这些内分泌活性植物化学物质与脊椎动物内分泌系统相互作用仍不清楚2，4。
植物雌激素，研究最充分的内分泌活性植物化学物质，是多酚类PSM，在结构和功能上模仿雌激素，直接与脊椎动物内分泌系统5的假体-垂体淋巴轴相互作用。在人类饮食中摄入植物雌激素与预防某些癌症、心脏病和更年期症状有关，尽管其他影响包括生育问题。事实上，这些化合物的生理影响是在20世纪40年代发现的，当时绵羊的不育归因于它们对富含植物雌激素的三叶草（三叶草）的放牧。摄入后，植物雌激素可以进入细胞并模仿雌激素的作用。虽然植物雌激素对绵羊的生育能力有负面影响，但植物雌激素与生理学之间的关系并不简单。与绵羊一样，南方白犀牛对源自大量大豆和紫花花的饲料中的雌激素化合物表现出敏感性。在怀孕期间喂养这种饮食的女性的女儿不太可能繁殖7。然而，其他研究表明，植物雌激素也可能有积极的影响，包括卵巢卵泡的成熟在老老鼠8，预防某些癌症，抗氧化活性和抗增殖作用9。
植物雌激素的影响范围并不奇怪，因为雌激素影响广泛的生物功能，包括生长，发育和调节生殖和中枢神经系统10。虽然有许多作用机制，植物雌激素往往有能力修改，增强或破坏雌激素信号，通过他们的能力，作为配体的核内雌激素受体阿尔法和β（ER+和ER+）。许多植物雌激素具有类似于雌激素的酚环结构，允许它们结合雌激素受体。那些具有痛苦的雌激素活性功能，如雌激素，形成一个激活的ER-ligand复合物，可以变暗和结合雌激素反应元素（ERE），并触发基因转录11。因此，雌激素和植物雌激素通过它们作为转录因子的行为来调节细胞活动和系统功能。
在这里，我们介绍了一种新颖的利用基于细胞的检测方法来筛选植物提取物，以发现具有雌激素生物活性的化合物的存在。这种检测使用中国仓鼠卵巢CHO细胞设计，以高度表达ER+，已被转染与萤火热病毒（Photinus皮拉里斯）荧光酶基因链接到ERE促进者12。当雌激素化合物存在时，它们与急ER结合，变暗，并结合到ERE，导致荧光酶基因的转录。加入基板溶液后，卢西费酶催化导致光子发射的反应。因此，阳性样品产生的光和阴性样品不。
这种市售的检测无需实验室用报告基因和雌激素受体13、14来传染哺乳动物细胞，这些受体的功效不稳定且变化无常。该检测提供了一个稳定的转染平台，能够快速而简单地通过受体结合确定植物是否具有雌激素活性。
我们测试了这样一个假设，即大豆的雌激素活性高于所有其他食物，因为大豆已知浓度为雌激素异黄酮15， 使用来自当地杂货店的人类食物。

<table><tbody><tr><td>1000 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>20 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176; water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176;, humidified 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>analytical balance</td><td></td><td></td><td></td></tr><tr><td>cell culture-rated laminar flow hood</td><td></td><td></td><td></td></tr><tr><td>dimethyl sulfoxide</td><td></td><td></td><td></td></tr><tr><td>disposable media basin, sterile</td><td></td><td></td><td></td></tr><tr><td>drip filtration system</td><td></td><td></td><td></td></tr><tr><td>Erlenmeyer flasks</td><td></td><td></td><td>125 mL and 250 mL</td></tr><tr><td>HPLC grade methanol</td><td></td><td></td><td></td></tr><tr><td>Human ER&amp;beta; Reporter Assay System, 1 x 96-well format assays</td><td>Indigo Biosciences</td><td>IB00411</td><td>Assay kit - analyzes 24 samples plus standard curve</td></tr><tr><td>lyophilizer</td><td></td><td></td><td></td></tr><tr><td>multi-channel pipette</td><td></td><td></td><td></td></tr><tr><td>orbital shaker</td><td></td><td></td><td></td></tr><tr><td>plate-reading luminometer</td><td></td><td></td><td>ex. Bioteck Synergy HTX</td></tr><tr><td>rotory evaporator</td><td></td><td></td><td></td></tr><tr><td>round bottom flasks</td><td></td><td></td><td>50 mL and 300 mL</td></tr><tr><td>sterile microcentrifuge tubes or sterile multi-channel media basins</td><td></td><td></td><td></td></tr><tr><td>sterile tips</td><td></td><td></td><td>200 &amp;micro;L and 1000 &amp;micro;L</td></tr><tr><td>Whatman grade 1 paper</td><td></td><td></td><td></td></tr><tr><td>whirl-pak bags</td><td></td><td></td><td>sterile polyethylene bags</td></tr></tbody></table>

screening for phytoestrogens using a cell-based estrogen receptor  &#946; reporter assay

植物是许多动物的食物来源，它们可以产生成千上万的化学物质。其中一些化合物影响消耗它们的脊椎动物的生理过程，如内分泌功能。植物雌激素，研究最充分的内分泌活性植物化学物质，直接与脊椎动物内分泌系统的下丘脑-皮质淋巴轴相互作用。在这里，我们介绍了一种新颖的利用基于细胞的检测方法来筛选植物提取物，以发现具有雌激素生物活性的化合物的存在。这种分析使用哺乳动物细胞设计，以高度表达雌激素受体β（ER+），并且已经与露西费酶基因转染。接触具有雌激素活性的化合物会导致细胞产生光。这种检测是测试生物雌激素活性的可靠和简单方法。它比瞬态透变检测有几种改进，最明显的是易于使用、细胞的稳定性和检测的灵敏度。

在人类饮食中常见的22种水果和蔬菜提取物被筛选出雌激素化合物的存在。各种食物被检测，包括豆类，如大豆，雪豌豆和豌豆，因为豌豆家族是已知的植物雌激素16的来源，以及无花果，枣，玉米，胡萝卜，苹果，香蕉，草莓，番茄，甘蓝和卷心菜。内分泌破坏化合物存在于常见物质（例如塑料和杀虫剂）中，有些通过红外体17具有生物活性。如果可能，对有?...

Watch this Scientific Journal Video about Screening for Phytoestrogens using a Cell-based Estrogen Receptor  Î² Reporter Assay at JoVE.com

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

我们优化了一种市售雌激素受体β记者检测，用于筛查人类和非人类灵长类动物的食物，以进行雌激素活性活动。我们通过显示已知的雌激素人类食物大豆注册高，而其他食物显示没有活性来验证这一分析。

使用基于细胞的雌激素受体对植物雌激素进行筛查β记者分析

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

screening-for-phytoestrogens-using-cell-based-estrogen-receptor

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

4.8K 次观看.  Indiana University. 我们优化了一种市售雌激素受体β记者检测，用于筛查人类和非人类灵长类动物的食物，以进行雌激素活性活动。我们通过显示已知的雌激素人类食物大豆注册高，而其他食物显示没有活性来验证这一分析。

视频: Screening for Phytoestrogens using a Cell-based Estrogen Receptor  β Reporter Assay

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

科研

环境科学

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

在用水筛查内分泌活动市售的<em&gt;体外</em&gt;式激活生物测定

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

利用哺乳动物双杂交法检测雌激素受体α的配联域二维化活性

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

癌症研究

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

用于筛查绝经后心血管功能障碍的外源雌激素治疗的活体雌激素缺乏小鼠模型

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

生物化学

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

单分子鱼的转录活性等位基因单细胞分析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

医学

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

使用 16α-[18F]-氟-17β-雌二醇 PET 可视化雌性卵巢切除小鼠人乳腺癌异种移植物中雌激素受体α表达

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

乳腺癌代谢调控的系统生物学雌激素受体信号

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

生物学

Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity

Source: Fender, E. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61005">Screening for Phytoestrogens using a Cell-based Estrogen Receptor &#946; Reporter Assay.</a> J. Vis. Exp. (2021).
This video describes a cell-based reporter assay to determine the estrogenic activity of plant-derived compounds. A compound with estrogen-like activity mimics its binding to estrogen receptors and drives downstream reporter enzyme gene transcription. The reporter enzyme generates bioluminescence in a reaction, indicating the estrogenic activity of the compound.

雌激素受体报告基因活性检测筛选植物雌激素雌激素活性

Source: Fender, E. et al., Screening for Phytoestrogens using a Cell-based Estrogen Receptor &#946; Reporter Assay. J. Vis. Exp. (2021).
This video ...