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?...

<ol>
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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?...

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 border="0" cellpadding="0" cellspacing="0" style="width:721px;" width="722">
	<colgroup>
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1000 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">37 &#8451; water bath</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">37 &#8451;, humidified 5% CO2 incubator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% ethanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">analytical balance</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">cell culture-rated laminar flow hood</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">dimethyl sulfoxide</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">disposable media basin, sterile</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">drip filtration system</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Erlenmeyer flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>125 mL and 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HPLC grade methanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Human ER&#946; Reporter Assay System, 1 x 96-well format assays</td>
			<td style="width:80px;">Indigo Biosciences</td>
			<td style="width:119px;">IB00411</td>
			<td>Assay kit - analyzes 24 samples plus standard curve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">lyophilizer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">multi-channel pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">orbital shaker</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">plate-reading luminometer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>ex. Bioteck Synergy HTX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">rotory evaporator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">round bottom flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>50 mL and 300 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">sterile microcentrifuge tubes or sterile multi-channel media basins</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sterile tips</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>200 &#181;L and 1000 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Whatman grade 1 paper</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">whirl-pak bags</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></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 ...

We use this assay to assess whether plant foods contain phytosteriods, which can affect physiological processes in animals, as well as behavior and reproductive success. This assay provides a simple and reliable method for determining whether plants produce estrogenic compounds with biological activity. This method was developed initially to screen drugs with xenoestrogen activity for applications in medicine.For example, inhibiting cell proliferation. We are using it to provide insight into primate ecology and evolution. It is important to have practice working with aseptic technique in a clean environment prior to attempting this assay, as it is very sensitive to contamination.Begin by freeze-drying the fresh plant items with the lyophilizer, then finely grind them using a grinding mill with a 0.85 millimeter mesh screen. Store the ground samples in bags in the dark until extraction. To extract any potential phytosteroids in the plants, add dried sample to an appropriately sized flask, then at HPLC grade methanol.Cover the plant methanol solution with aluminum foil and set it to rotate at 100 RPM for three days on an orbital shaker. After three days, use filter paper to decant the supernate and into a drip filtration system. Dry the plant extract with a rotary evaporator until it is thickened, but pourable in a 300 milliliter round bottom flask.Pour the sample into a 50 milliliter flask and rinse the large flask with a small amount of methanol, then continue to dry the sample in the small flask until the methanol is completely evaporated. When finished, weigh the sample residue with an analytical balance and record the mass. Dissolve the plant extract in DMSO at a concentration of 0.1 gram of extract to two milliliters of DMSO and vortex it until homogenized.DMSO is not toxic on its own, but it is a vehicle that allows other substances to cross membranes. Since we do not know if our plants contain toxic chemicals, all are treated as if they're hazardous. Do not allow the solution to touch skin, replace gloves if contaminated, and wear thick, close-toed shoes and lab coats.Store the sample at four degrees Celsius in amber glass vials until ready to use. Vortex the samples, then add four microliters of each sample to 496 microliters of compound screening medium to yield a 0.8%DMSO solution. Disinfect the outside surface of a pre-warmed cell recovery medium tube with 70%ethanol and transfer 10 milliliters of the medium into a tube of frozen reporter cells to thaw them.Close the tube of reporter cells and place it in a 37 degrees Celsius water bath for five to 10 minutes. After retrieving the cells from the water bath, gently invert the tube several times to break up aggregates of cells and produce a homogenous suspension, then clean the surface of the tube with 70%ethanol. Use a multi-channel pipette to dispense 100 microliters of the reporter cell suspension into each well of a 96 well plate, then dispense 100 microliters of samples in triplicate into the appropriate wells.Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 22 to 24 hours. Just prior to the end of the plate incubation remove detection substrate and detection buffer from the refrigerator and place them in a low light area until equilibrated to room temperature, then gently invert each tube to mix the solutions. Immediately before the plate incubation is complete, pour the entire contents of the detection buffer into the tube of detection substrate to create luciferase detection reagent.Mix the tube gently so as not to produce foam. Discard the contents of the sample plate into inappropriate waste container and gently tap it on a clean absorbent paper towel to remove the last droplets from the wells. Add 100 microliters of the luciferase detection reagent to each well and allow the assay plate to rest at room temperature for 15 minutes, then quantify the luminescence using a 96 well plate reading luminometer.22 extracts of fruits and vegetables commonly found in human diets were screened for the presence of estrogenic compounds. Estrogenic activity is presented in an ordinal qualitative manner of high, medium, low, or no activity. Organic and non-organic soybeans screened at high levels of activity, while all other fruit and vegetable items registered no activity.Comparing soybean results to the standard curve also shows that they have high estrogenic activity levels at this concentration. Soybean extract, a known potent source of the isoflavones, daidzein and genistein, was further used to determine the dilution yielding a 50%signal to the maximum. This extract requires 422 times more dilution to produce half the signal of our standard dilution protocol.Samples identified with activity could be sent for analysis by mass spectrometry to identify the active chemicals. Using an antagonist method, we could also determine if the plant food contains chemicals that reduce the binding and activity of endogenous estrogens. This technique allows us to further our understanding of how prevalent estrogenic foods are in primate diets, as well as how consumption of phytoestrogens affects physiology and behavior when the assay is combined with fecal hormone analyses and field data.

Research

JoVE Journal

Biochemistry

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

使用新型不溶性生色底物检测试剂盒碳水化合物降解酶的高通量筛选

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

科研

生物化学

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

细菌内膜显示筛查抗体片段的图书馆

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

An In Vivo Estrogen Deficiency Mouse Model 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 ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

使用核酸探针进行核酸活性的动力学筛选

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

使用 PRESTO-Tango Assay 在 GPCR-宽范围范围内对 β-Arrestin2 进行配制筛查的平行审讯

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

基于核磁共振的片段筛选，样品最少，自动化程度最高

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...