Name: gap junctional intercellular communication: a functional biomarker to assess adverse effects of toxicants and toxins, and health benefits of natural products
Uploaded: 2016-12-25T13:00:00
Description: Watch this Scientific Journal Video about Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Supported by NIEHS grants #R01 ES013268-01A2, and the contents are solely the responsibility of the author and do not necessarily represent the official views of the NIEHS, and supported by CETOCOEN UPgrade project No. CZ.1.05/2.1.00/19.0382.

这项研究的方案经动物护理和日本健康科学，这就是体内都做过实验，全国学院利用委员会，以确保将大鼠人道和对苦难的缓解方面的治疗。 1. SL-DT生物测定<ol><li>种子3×10 5个 WB-F344大鼠肝脏上皮细胞到35 mm直径培养含有鹰板改性介质加5％牛胎儿血清，并培养所述细胞在培养箱中在37ºC，100％相对湿度（RH），5％ 的 CO 2。 </li><li>培养细胞，直到它们达到100％?...

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对SL-DT测定是在测定GJIC简单和通用的技术，但也有要在设计适当的实验方案来解释几个关键的关注。对于使用SL-DT测定细胞间隙连接通讯的强大的测量必须有通过细胞间隙连接的LY良好的染料扩散。在最低限度，有足够的时间应选择以保证染料通过八个或更多列细胞扩展从两个方向的手术刀加载细胞。另外，为了便于将染料从手术刀装载的细胞迁移的距离的测量，选择确保了控制板的染料扩散填充...

该方法的总的目标是提供一种简单的，全面和相对廉价的技术来评估化合物的潜在毒性。这是一种体外方法，可以在多种细胞系中使用。配备有落射荧光显微镜标准细胞生物学实验室可以通过此法进行研究。 我们的细胞功能的基本知识已经高度依赖于在体外生物测定，并已成为医药品，环境污染物，以及食品生污染物的毒理学评估的必要成分。不幸的是，没有一个统一的体外生物测定系统，能够全面地满足所有毒理学评估的要求。许多体外测定的设计和优化，以评估以及评估一个特定的生物化学或分子端点。这些经常以高通量设置以反映一个扰动组合特定信号转导途径，如雌激素受体信号1。这一策略相当成功，但参与基因表达的信号转导通路的数量繁多，因此选择一个特定的信号通路，以评估相当复杂的任务。高通量的协议，目前正在开发的，并用于同时测量许多信号传导途径，这一直是一个方法克服一些单个分析的局限性。然而，并非所有的信号通路已经成功掺入更全面的办法，再加上新的信号传导途径正在不断地被发现，进一步复杂化这个评估过程。使用的体外方法广泛数字，特别是高通量系统中，综合毒理学评估也非常昂贵，并且不利于大多数单研究者领导的研究项目。 GJIC是一个过程TIG通过在电压变化，钙离子浓度，pH值，氧化还原平衡，由主要的细胞内信号转导途径和相互作用与膜和细胞骨架蛋白2,3-调节htly控制。因此，GJIC的抑制可以反映不同类型的细胞应激，不同的细胞功能的破坏，或不同的信号转导途径的扰动。克服利用有限的信号转导的生物测定的另一种方法是采取的生物现象优点，即许多，如果不是大多数，信号转导途径被进一步通过合作的细胞间的信号系统，通过间隙连接通道4-8调制。虽然间信号系统也很多，根据多路径控制，通过间隙连接通道间信号是最终被打开的通道的功能，部分封闭或完全封闭。这提供了可以使用各种可容易地测定的端点在体外生物分析系统7。考虑到组织的稳态设定点，需要畅通的渠道，确定化合物对间隙连接通讯的影响（GJIC）在确定化合物4,8的潜在的毒性作用更全面的方法。在本质上，在协调多个信号转导事件控制基因表达的核心作用这一关键的生物现象，使得毒性作用广泛评估。因此，评估GJIC生物测定是一个很好的起点，以评估化合物的潜在毒性。 用于评估GJIC最广泛的技术是基于预加载的细胞用荧光探针，然后从所装载的细胞或细胞相邻小区监控染料的迁移。技术预加载染料都涉及显微注射9，刮去探头11装载10和甲基酯</suP&gt;。手术刀装载荧光染料转移（SL-DT）的方法是刮负载的修改-染料埃尔Fouly 10开发的传输试验。而不是更微创刮，本报告的手术刀加载方法包括通过细胞减少侵入破坏（ 图1）单层圆刀片手术刀的温柔卷。这种技术的优点是细胞而非显微注射法的单细胞的群体的毒理学评估。此外，该测定的简单性允许在短时间内快速检测多块板而使用显微注射技术以及使用荧光探针的甲酯技术方法都是显著更费时，并且需要相当高的技术水平。 虽然没有单一的方法来满足研究细胞间隙连接通讯的所有需求;在SL-DT分析是一种简单的，相当便宜，多才多艺的分析，可以满足多种需求的各种化合物毒性的初步评估。主要优点包括：简单，设备或技能没有特殊需要所需要的其他方法，如微注射，荧光漂白恢复（FRAP）分析和分子荧光探针测定法，GJIC的在一个大一个快速和同时评估的局部激活后细胞的数目，有利于高通量设置用自动化荧光显微镜成像和分析，以及其适应性对于体内研究。

<table border="0" cellpadding="0" cellspacing="0" width="1238">
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			<td height="63" style="height:63px;width:312px;">WB-F344 rat liver epithelial cells</td>
			<td style="width:217px;">From Drs. J. W. Grisham and M. S. Tsao of the University of North Carolina (Chapel Hill, NC)</td>
			<td style="width:279px;">none</td>
			<td style="width:431px;">Provided by Drs. J. W. Grisham and M. S. Tsao University of North Carolina-Chapel Hill-NC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">35 mm Culture Plates</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">25 cm2 culture flasks</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">75 cm2 culture flasks</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-medium, an Eagles modified medium&#160;</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">Formula No. 78-5470EF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">fetal bovine serum</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">10437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% trypsin-EDTA&#160;</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">15050</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">phosphate buffered saline</td>
			<td style="width:217px;">homemade</td>
			<td style="width:279px;">see below for ingredient cat#&#39;s</td>
			<td>137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 2 mM KH2PO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">KCl</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3040-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NaCl</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3624-05</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3819--01</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lucifer Yellow CH, lithium salt</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">L0259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">rhodamine-dextran</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">R9379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1-methylanthracene</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">phenanthrene</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">P11409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">resveratrol</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">R5010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">D609</td>
			<td>Tocris Bioscience&#160;</td>
			<td style="width:279px;">1437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">acetonitril</td>
			<td>EMD</td>
			<td style="width:279px;">AX0145-1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:312px;">37% solution formaldehyde</td>
			<td>JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">2106-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">#20 surgical blade</td>
			<td>Fine Science Tools Inc.&#160;</td>
			<td>10317-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">50 mL conical sterile tubes</td>
			<td>Thermo scientific&#160;</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon epifluorescence microscope&#160;</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">Eclipse TE300</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Nikon FITC dichroic cube</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">96107</td>
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			<td height="21" style="height:21px;">&#160;CCD camera&#160;</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">Nikon Cool Snap EZ CCD</td>
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			<td height="21" style="height:21px;">&#160;imaging system.</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td>Nikon NIS-Elements F2.2 imaging system.</td>
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			<td height="21" style="height:21px;width:312px;">Image J</td>
			<td style="width:217px;">National Institute of Health</td>
			<td style="width:279px;">http://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
	</tbody>
</table>

gap junctional intercellular communication: a functional biomarker to assess adverse effects of toxicants and toxins, and health benefits of natural products

这个协议描述，其测量通过间隙连接通道间通讯，这是一个主要间过程，其中组织稳态保持手术刀放入荧光染料转移（SL-DT）技术。由毒物，毒素，药物等间隙连接通讯（GJIC）的中断已被链接到大量的不良健康影响。许多基于遗传人类疾病都与在间隙连接的基因突变。在SL-DT技术是GJIC在一个人口众多细胞的同时评估一个简单的功能测定。该测定通过细胞群体简要扰动细胞膜用手术刀刀片涉及预加载的细胞用荧光染料。然后荧光染料允许间隙连接通道横穿通过对相邻小区一段指定的时间。该试验然后通过加入福尔马林至细胞终止。在萤光灯的传播通过细胞群体rescent染料评估用落射荧光显微镜和图像被任何数量的形态软件包可用，包括在公共领域发现自由软件包进行分析。此法也适用于使用组织切片从处理过的动物各器官的体内研究。总体而言，对SL-DT测定可以起到广泛的体外药理学和毒理学的需要，并且可以潜在地适于使用自动荧光显微镜成像和分析的高通量设置系统在较短的时间以阐明更多的样品。

GJIC中断已被广泛用作生物标记物用于鉴定处于导致不利的健康影响14基因控制的nongenotoxic，后生水平的有毒化合物。例如，多环芳香烃（PAHs）是环境的无所不在的污染物，但在他们的后生毒性作为其分子结构15的函数而变化。较低分子量多环芳烃通常发现在相对较高的浓度比在许多不同的环境中的更高分子量的多环芳烃如被污染的河流沉积物到该香烟烟?...

Watch this Scientific Journal Video about Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products 

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

本协议描述，通过间隙连接通道测量细胞间通讯手术刀装载荧光染料转移技术。间隙连接通讯是一个主要的细胞过程，其中组织稳态维持这种细胞信号中断了不良的健康影响。

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

Cell-Cell Interactions

SCIENTISTS IN ACTION

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

使用不固定，冷冻的组织，以研究自然的粘蛋白分布

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

科研

生物学

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

使用MALDI-TOF质谱和自定义数据库的表征细菌土著一个独特的洞穴环境（卡切内岩洞，AZ，USA）

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

隔离和线粒体从培养细胞和小鼠组织功能分析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

评估细菌素对小鼠肠道微生物的影响的方法

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

一种碘-黄色荧光荧光蛋白-缝隙--细胞间通信方法

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

风险蝴蝶捕获繁殖计划，以提高生命历史知识和有效的异地保护技术

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Field Identification of Matricaria chamomilla using a Portable qPCR System

使用便携式 qPCR 系统对 马特里卡里亚查莫米拉进行现场识别

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

在靶标和标记下使用裂解分析植物中的H3K4me3修饰

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...