Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

这项工作得到了韩国粮食、农业和林业技术规划和评价研究所 (ipet) 通过农业、粮食和农村事务部资助的农业-生物工业技术发展方案 (mafra) 的支持。) (11159-02-wt011) 和韩国大学 bk21 plus 生命科学和生物技术学院。

1. 培养基、化合物和试剂的制备
<ol><li>准备 b16f10 生长完整的介质。补充 dulbecco 的改良鹰培养基 (dmem) 与10% 的胎牛血清 (fbs) 和1% 的青霉素/链霉素 (pest)。<ol>
<li>要使500毫升的完整介质, 将 dulbecco 改性的 eagle 介质 (dmem) 的445毫升与50毫升的 fbs 和5毫升的 pest 混合在一个1升的灭菌玻璃瓶中。轻轻混合后, 通过0.2μm 的瓶盖过滤器将介质过滤到新的灭菌玻璃瓶中, 然后存放在4°c。 注意: 所有细胞培养?...

<ol>
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我们提出了评估使用培养的黑素细胞的测试化合物的低细胞化活性的方案。有代表性的结果显示了抗酪氨酸酶抑制剂 arbutin 的低细胞化作用, 它抑制酪氨酸酶活性和细胞黑色素含量。这些方法在抗黑色素活性研究中得到了广泛的应用。利用这些检测方法, 我们还成功地发现了几种生物活性化合物, 这些化合物在过去十年中对b16f10 细胞产生黑色素作用, 9、10</sup...

黑色素在包括皮肤、眼睛和大脑1在内的几个器官的生理、病理和毒理学中发挥着至关重要的作用。黑色素的主要功能是光筛选和生化效应。黑色素吸收近红外光和可见光以及紫外线 (uv) 辐射, 在较短波长的光的情况下吸收率提高;因此, 黑色素保护组织免受可见光或紫外线辐射2造成的损害.黑色素是一种抗氧化剂, 对金属和其他有毒化学品有亲和力;因此, 它可以保护组织免受氧化和化学应激3。然而, 黑色素的过度生产会导致皮肤病问题。
皮肤和虹膜中黑色素的数量和质量是虹膜和皮肤颜色的最重要决定因素。个人可能有不同的肤色偏好;有的喜欢晒黑的皮肤, 有的则喜欢较浅的皮肤颜色。根据这些消费者的情况, 低菌化妆品已经开发, 以满足个别市场的青睐的肤色4。因此, 研究低细胞和抗黑色素活性在科学和实践上都很重要。
黑色素生成是黑色素通过一系列酶性和自发化学反应在黑色素细胞中进行生物合成的复杂过程。一个黑素细胞被大约36个角质形成细胞包围, 黑素细胞是黑色素合成工厂, 将它们的产品分配给邻近的角质形成细胞。在皮肤中, 产生和储存在黑色素细胞黑色素瘤室中的黑色素通过树突输送到表皮中的角质形成细胞. 
l-tyrosine 作为黑色素生成的初始底物, 酪氨酸酶催化两种连续反应, 将 l-酪氨酸转化为 3, 4-二羟基苯基丙氨酸 (dopa), 然后转化为多巴米酮。这些反应是黑色素生成的限速步骤 5,6。因此, 低细胞分裂活性可以通过直接评估细胞酪氨酸酶活性来测量。为此, 含有酪氨酸酶的黑素细胞提取物由 dopa 孵育, 样品中产生的多巴西酮可在475纳米的分光光度法中进行测定。这些值通过样品的蛋白质浓度进行归一化, 与对照相比, 具有低渗活性的物质会导致多巴奎因的形成较少。
其次, 低细胞分裂活性可以通过直接测量培养的黑色素细胞中的黑色素来量化。用试验材料处理细胞后, 在碱性条件下提取黑色素, 用分光光度法在400纳米处测定黑色素含量。低细胞化剂会导致比对照组7更低的黑色素含量。
最后, 利用 Fontana-Masson 黑色素染色和随后的图像分析可以量化低细胞分裂活性。在 Fontana-Masson 染色中, 黑色素颗粒将硝酸铵还原为可见的黑色金属状态, 而微观图像中的黑细胞区域代表黑色素的数量。
低渗剂通常会给出与这三种方法一致和可比的结果, 这证实了该物质的活性是有效的。或者, 它可能是有用的, 以衡量关键基因和蛋白质在黑色素生成的表达, 以响应测试物质, 以检查低细胞化活性。除了酪氨酸酶外, 酪氨酸酶相关蛋白 (trp-1) 和多巴斯姆自体擦除 (trp-2) 是黑色素生成的关键酶 8.转录因子、微邻苯二甲相关转录因子 (mitf) 是黑色素生成和基因蛋白表达水平定量的主要调节剂, 也可用于评估低细胞化活性。

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

本研究提出了体外低细胞化活性定量的实验室方法。黑色素是黑色素细胞中的主要色素, 是针对多种细胞和环境因素而合成的。黑色素保护皮肤细胞免受紫外线伤害, 但也具有生物物理和生化功能。黑色素细胞的过度产生或积累会导致皮肤病问题, 如雀斑、黑斑、黄瘤和痣。因此, 控制黑色素生成与低渗剂是重要的个人有临床或美容需求。黑色素主要是在黑色素细胞的黑色素体中合成的, 这个过程被称为黑色素生成, 它受到外部和内在因素的影响, 如激素、炎症、年龄和紫外线照射。我们描述了三种方法来确定化学物质或天然物质在黑素细胞中的低细胞化活性: 测量 1) 细胞酪氨酸酶活性和 2) 黑色素含量, 3) 染色和量化细胞黑色素与图像分析。
在黑色素生成过程中, 酪氨酸酶催化将 l-酪氨酸转化为 3, 4-二羟基苯基丙氨酸 (l-dopa), 然后转化为多巴基宁的限速步骤。因此, 抑制酪氨酸酶是一种主要的低细胞化机制。在培养的黑素细胞中, 通过添加 l-dopa 作为底物, 用分光光度法测定多巴西酮的产生, 可以量化酪氨酸酶活性。黑色素生成也可以通过定量测量黑色素含量。用 naoh 提取含黑色素的细胞分数, 用定量分光光度法提取黑色素。最后, 黑色素的图像分析可以量化黑色素的含量。虽然这些体外检测的结果可能并不总是在人体皮肤中复制, 但这些方法在黑色素生成研究中被广泛使用, 特别是作为识别潜在低细胞化活性的第一步。这些方法也可用于评估黑素细胞的活性、生长和分化。与三种不同方法一致的结果确保了效果的有效性。

具有代表性的结果, 在 b16f10 黑色素细胞的抗黑色素素化合物的低细胞分裂活性如下所示。图 1a显示, 与车辆处理的对照相比, arbutin 显著抑制了细胞酪氨酸酶活性。同样, 与对照组相比, 阿布丁刺激的细胞黑色素含量显著降低 (图 1b)。用 Fontana-Masson 染色染色的细胞的显微图像如图 1c所示。与对照相比, arbutin ...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

我们描述了三种实验方法来评估化学物质在体外的低细胞化活性: 1) 细胞酪氨酸酶活性和 2) 黑色素含量的定量, 以及 3) 细胞黑色素染色和图像分析测量黑色素。

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

该方法有助于开发具有抗黑色致病活动的功能性化妆品和皮肤剂。它是有执行和结果可以迅速获得。此外，这种方法可能有用的研究人员，谁希望确定或调查的影响或化合物或研究抗梅拉诺原或亲源性活动。测量酪氨酸酶活性，首先在细胞培养箱中24小时以完整培养基箱中，以每24个井板浓度的5倍10至第四细胞播种B16-F10黑色素杀菌剂。第二天，用DMEM更换每井的上能，辅以新鲜准备的测试化合物和抑制剂控制或阴性控制，将板再返回培养箱72小时。在治疗结束时，每口井用300微升的冷 pbs 冲洗油井两次，将盘子放在冰上。接下来，使用细胞刮刀在五分钟后收集细胞解液，将300微升的解解缓冲液添加到每井中。将产生的细胞颗粒悬浮液转移到单个 1.5 毫升微离心管中。在冰上以14，500转/小时将酸盐均质三次，两秒钟，以释放细胞间酪氨酸酶。通过离心将细胞碎屑进行分离，将超热剂转移到冰上单个1.5毫升离心管。将每个上清液的 70 微升分配到透明 96 孔聚苯乙烯微板的单个孔中，并将 140 微升的 Tyrosinase 基板溶液添加到每口上清液孔中。轻轻摇动盘子，混合井内。然后在37摄氏度下孵育板两小时。并在微孔板读卡器上测量 475 纳米的酪氨酸酶活性。测量细胞的黑色素含量后收集处理细胞的 lysate 显示。通过离心收集酸盐，将中流剂转移到新管中。在每个颗粒中加入300个普通氢氧化钠的300微升，在60摄氏度下孵育一小时。其次是溶解细胞悬浮的离心。然后将200微升的超糖转移到96井板的每个井中。以 400 纳米波长测量微孔板读取器上的黑色素含量。对于Fondna-Masson染色，用300微升的冷pbs清洗两次经过处理的细胞，用200微升10%的甲醛在4摄氏度下固定一小时。用蒸馏水冲洗厚细胞，并在每口井中加入200微升58至68摄氏度的氨化银工作溶液。在37摄氏度下孵育一小时，或直到细胞变成黄褐色。用蒸馏水冲洗染色细胞，随后在室温下以0.1%的氯化金孵育两分钟。用蒸馏水冲洗氯化金处理细胞，向细胞中加入200微升硫化钠溶液。两分钟后再次冲洗细胞。每井用200微升的核快红标记细胞5分钟。然后在光显微镜下成像之前，先用自来水清洗染色的细胞。对于阈值分析，请打开图像 J 中的图像并选择图像、调整和颜色阈值。要测量沾染丰塔纳马松的区域，请将亮度参数栏设置为零，并调整亮度到包含所有黑色或棕色染色细胞的点。选择"分析和分析粒子"并选中"分析粒子"窗口中的汇总框，然后单击"确定"以获取结果摘要，然后将数据复制并粘贴到电子表格中。与控制车辆处理的细胞相比，阿布丁治疗显著抑制细胞酪氨酸酶活性。同样，与对控组相比，使用阿布丁刺激的细胞的黑色素含量显著降低。虽然治疗组之间的细胞在形态上相似，但与对照处理的细胞相比，Arbutin会降低黑色色素的面积。此方法可用于使用我们的复合库进行活动筛选。有数百个样品需要快速测试。此外，此方法还可用于研究涉及黑色素发生的机制。在生物活性候选化合物被确定后，为研究生物机制或商业应用提供了证据。

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

的pH依赖性活性的检测<em&gt;大肠杆菌</em&gt;陪护HdeB<em&gt;体外</em&gt;和<em&gt;在体内</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

科研

生物化学

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

丰度和收费水平的转移 Rna 在大肠杆菌中的量化

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

映射代谢: 直接在组织中监测乳酸脱氢酶活性

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

体外检测 tRNA-Isopentenyl 转移酶活性的实验研究

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

蜜蜂匀浆中磷脂酶 C 活性的检测

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...