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.

Protokollen for denne undersøgelse blev godkendt af Animal Care og Udnyttelse udvalg af National Institutes of Health Sciences i Japan, hvilket er hvor in vivo forsøg blev gjort, for at sikre, at rotterne blev behandlet humant og med hensyn til lindring af lidelse. 1. SL-DT Bioassay <ol><li> Seed 3 x 10 5 WB-F344 rottelever epitelceller onto 35 mm diameter dyrkningsplader indeholdende Eagles modificeret medium plus 5% føtalt bovint serum, og dyrke cellerne i en inkubator ved 37 ºC, 100%...

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SL-DT-analysen er en enkel og alsidig teknik måling GJIC, men der er flere kritiske bekymringer, der skal bogføres i at designe passende eksperimentelle protokoller. For robuste målinger af GJIC anvender SL-DT-assay skal der være et godt farvestof spredning af LY gennem gap junctions af cellerne. Som minimum bør vælges en passende tid til at sikre, at farvestoffet spredes gennem otte eller flere rækker af celler fra skalpellen indlæst celler i begge retninger. Også for den lette målingen af ​​afstanden, at...

Det overordnede mål med denne fremgangsmåde er at tilvejebringe en enkel, omfattende og forholdsvis billig teknik til at vurdere de potentielle toksiciteter af forbindelser. Dette er en in vitro fremgangsmåde, der kan anvendes i multiple cellelinier. Standard cellebiologiske laboratorier udstyret med epifluorescens mikroskoper kan foretage målinger ved hjælp af denne analyse. Vores grundlæggende viden om cellefunktioner har været meget afhængig af in vitro-bioassays, og er blevet en væsentlig komponent i toksikologiske vurderinger af lægemidler, miljøforurening, og fødevarer født forureninger. Desværre er der ingen enkelt in vitro bioassay system, omfattende kan opfylde kravene til alle toksikologiske vurderinger. Mange in vitro-assays er udformet og optimeret til at evaluere samt vurdere en specifik biokemisk eller molekylær endepunkt. Disse er ofte kombineret i et højt gennemløb sat op til at afspejle en forstyrrelse af envisse signaltransduktionsvej, såsom østrogen-receptor signalering 1. Denne strategi har været ret vellykket, men det omfattende antal signaltransduktionsveje der er involveret i genekspression gør opgaven med at vælge en specifik signaleringsvej at vurdere temmelig komplekse. Høje gennemløb protokoller er under udvikling og anvendes til samtidigt at måle talrige signalveje, hvilket har været en fremgangsmåde for at eliminere nogle af begrænsningerne af enkelte assays. Imidlertid har ikke alle signalveje blevet indarbejdet i mere omfattende metoder, plus nye signalveje konstant bliver opdaget, at yderligere komplicerer denne vurdering proces. Brug omfattende antal af in vitro-metoder, særligt høje gennemgående sat systemer, for omfattende toksikologiske vurderinger er også meget dyrt og er ikke befordrende for de fleste enkelt investigator førte forskningsprojekter. GJIC er en proces tightly styret af ændringer i spænding, calcium koncentration, pH, redox balance, reguleret af de store intracellulære signaltransduktionsveje og interaktioner med membran og cytoskeleton proteiner 2,3. Således kan inhibering af GJIC afspejle forskellige typer af cellulært stress, forstyrrelser i forskellige cellulære funktioner, eller forstyrrelser af forskellige signaltransduktionsveje. En anden fremgangsmåde for at eliminere anvendelsen af begrænsede signaltransduktionsveje bioassays er at drage fordel af de biologiske fænomener, at mange, hvis ikke de fleste, signaltransduktionsveje er yderligere moduleret af kooperative intercellulære signalsystemer via gap junction kanaler 4-8. Selvom intercellulære signalsystemer er også talrige og under flere kontrol-biosyntesen, intercellulære signalering via gap junction-kanaler er i sidste ende en funktion af kanalerne åbnes, delvist lukket eller helt lukket. Dette tilvejebringer et endepunkt, der let kan måles ved anvendelse af forskelligein vitro bioassay-systemer 7. I betragtning af at den homøostatiske sæt punkt i et væv kræver åbne kanaler, bestemmelse af effekten af forbindelser på kløften junktion intercellulær kommunikation (GJIC) er en mere omfattende tilgang til at bestemme potentielle toksiske virkninger af forbindelser 4,8. I det væsentlige, denne kritiske biologiske fænomen, der spiller en central rolle i koordineringen af ​​flere signal transduktion begivenheder kontrollerer genekspression giver mulighed for en bred vurdering af toksiske virkninger. Således bioassays, der vurderer GJIC er et glimrende udgangspunkt for at vurdere toksiske potentiale af forbindelser. De mest omfattende teknikker anvendes til at vurdere GJIC er baseret på forbelastning celler med en fluorescerende probe og derefter overvåge migrationen af ​​farvestoffet fra den belastede celle eller celler til tilstødende celler. Teknikker forudindlæser farvestoffet har involveret mikroinjektion 9, skrabe loading 10, og methylestere af sonderne 11 </sup&gt;. Den skalpel lastning-fluorescerende farvestof overførsel (SL-DT) metode er en modifikation af skraber belastning - farvestof overførsel assay udviklet af El Fouly 10. Snarere end den mere invasive skrabe, skalpel loading fremgangsmåden ifølge denne rapport indebærer en blid rulle af en skalpel med en rund klinge gennem et monolag af celler, der minimerer invasive skader (figur 1). Fordelene ved denne teknik er den toksikologiske vurdering af en population af celler i stedet for enkelte celler af mikroinjektion assay. Endvidere enkelheden i dette assay tillader hurtig detektion af multiple plader på kort tid henviser metoder under anvendelse mikroinjektion teknikker og teknikker, der bruger methylestere af fluorescerende prober er betydeligt mere tidskrævende og kræver væsentligt højere færdighedsniveau. Selv om der ikke er nogen enkelt metode til at opfylde alle de behov, studere GJIC; SL-DT-assay er en simpel, forholdsvis billig og alsidig assay, der kanmøde mange af behovene for indledende vurderinger af toksicitet af forskellige forbindelser. Større fordele kan nævnes: enkelhed, ingen særlige behov for udstyr eller færdigheder, der kræves for andre metoder såsom mikroinjektion, fluorescerende bedring efter fotoblegning (FRAP) assay og lokal aktivering af molekylære fluorescerende probe assays, en hurtig og samtidig vurdering af GJIC i et stort antal celler, der bidrager til en høj produktion set-up med automatiseret fluorescens mikroskopi billedbehandling og analyse, samt dens tilpasningsevne til in vivo undersøgelser.

<table border="0" cellpadding="0" cellspacing="0" width="1238">
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	<tbody>
		<tr height="63">
			<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>
		</tr>
		<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>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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

Denne protokol beskriver en skalpel loading-fluorescerende farvestof overførsel (SL-DT) teknik, der måler intercellulær kommunikation via gap junction kanaler, som er et stort intercellulære proces, hvorved væv homeostase opretholdes. Afbrydelse af kløften junktion intercellulær kommunikation (GJIC) ved giftstoffer, toksiner, stoffer mv har været forbundet med en række sundhedsskadelige virkninger. Mange genetiske baserede humane sygdomme er blevet forbundet med mutationer i gap junction gener. SL-DT teknik er en simpel funktionel analyse til samtidig vurdering af GJIC i en stor population af celler. Assayet involverer forpåfyldning celler med et fluorescerende farvestof ved kortvarigt at forstyrre cellemembranen med en skalpelblad gennem en population af celler. Det fluorescerende farvestof lades derefter krydse gennem gap junction kanaler til naboceller for et defineret tidspunkt. Assayet afsluttes derefter ved tilsætning af formalin til cellerne. Udbredelsen af ​​fluospare- farvestof gennem en population af celler vurderes med en epifluorescensmikroskop og billederne analyseres med et vilkårligt antal morfometriske softwarepakker, der er tilgængelige, herunder gratis software pakker findes på det offentlige område. Dette assay er også blevet tilpasset til in vivo undersøgelser under anvendelse af vævssnit fra forskellige organer fra behandlede dyr. Alt i alt kan SL-DT assay tjene en bred vifte af in vitro-farmakologiske og toksikologiske behov, og kan potentielt tilpasses til high throughput set-up systemer med automatiseret fluorescens mikroskopi billedbehandling og analyse for at belyse flere prøver på kortere tid.

Afbrydelse af GJIC er blevet flittigt brugt som biomarkør til at identificere giftige stoffer på ikke genotoksisk, epigenetisk niveau af gen kontrol, inducerer sundhedsskadelige virkninger 14. For eksempel polycykliske aromatiske hydrocarboner (PAH) er allestedsnærværende miljøforurenende stoffer, men varierer i deres epigenetiske toksiciteter som en funktion af deres molekylære strukturer 15. De lavere molekylvægt PAH&#39;er findes typisk ved relativt højer...

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

Denne protokol beskriver en skalpel loading-fluorescerende farvestof transfer teknik, der måler intercellulær kommunikation via gap junction kanaler. Gap junktion intercellulære kommunikation er en væsentlig cellulær proces, hvorved væv homeostase opretholdes og afbrydelse af denne cellesignalering har sundhedsskadelige virkninger.

Gap junktion intercellulær kommunikation: En funktionel biomarkør at vurdere negative virkninger af giftstoffer og toksiner, og Sundhedsmæssige fordele af Naturprodukter

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

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