Name: a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals
Uploaded: 2018-06-25T12:30:00
Description: Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

Dette arbejde blev støttet af et pilotprojekt tilskud fra CREH center sponsoreret af NIEHS og National Natural Science Foundation of China (nr. 31572626).

Alle dyreforsøg blev henrettet i overensstemmelse med alle relevante retningslinjer, regler og reguleringsorganer. Protokollen demonstreres blev udført under vejledning og godkendelse af institutionelle Animal Care og brug udvalg (IACUC) af Texas A & M Institute for genomisk medicin.
1. løsning forberedelse
<ol><li>Fortynde 10 x Hank afbalanceret saltopløsning til 1 x med dobbelt destilleret H2O, og opbevares ved 4 ° C.</li>
	<li>Forbered isolation løsning ved at tilføje HE...

<ol>
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Akkumulere beviser tyder på, at eksponering for Miljøkontaminanter spiller en vigtig rolle i udviklingen af T2DM. Fremmedstoffer-induceret ROS er blevet anerkendt som en potentiel ætiologiske faktor, der bidrager til udviklingen af T2DM. Mennesker er udsat for en bred vifte af xenobiotiske kemikalier og der er et stort behov for nye forskningsteknikker effektivt identificere de pancreas giftstoffer og at efterforske mekanismen af toksicitet specifikke med cellerne i pancreas.
I denne unders...

Stigninger i prævalensen af T2DM er blevet en global sundhedskrise i de seneste år udgør en alvorlig trussel mod folkesundheden1. Mange faktorer er blevet fundet at være kausalt forbundet med udvikling af T2DM, blandt hvilke, tilbagevendende resultaterne tyder på at et fælles konvergerende point for disse faktorer er induktion af oxidativ stress, som fører til generation af overdreven ROS2 , 3.
Et bredt spektrum af Miljøkontaminanter herunder PCB, dioxiner og BaP har vist sig for at fremkalde oxidativ stress, som kan forringe funktionen af pancreas β celler, der fører til insulinresistens og T2DM4. Selv om den fysiologiske niveau af ROS spiller en vigtig rolle i cellulære funktioner, eksponering for ROS, som overstiger kapaciteten af antioxidant system resulterer i skader på celler/væv og fører til sygdomme5. Pancreas β celler udtrykker et lavt niveau af antioxidant enzym, og således er en følsom mål for oxidative stress-medieret skader6,7. Kronisk udsættelse for høje niveauer af ROS har vist sig at forårsage stress-induceret i bugspytkirtlen celle dysfunktion5 samt insulinresistens i lever og fedtvæv8.
Det overordnede mål med dette projekt er at udvikle en celle-baseret analyse for skærmen kemikalier for deres diabetogenic potentialer baseret på deres induktion af ROS i pancreas celler. Bugspytkirtlen mangler metaboliske afgiftning og er en følsom mål for xenobiotiske-induceret skade6,7. Derfor, ved direkte måling af den ROS, der er genereret i bugspytkirtlen celler, denne analyse bør give en direkte indbyrdes tilnærmelse af den kemiske-induceret skade i bugspytkirtlen. For at udvikle denne metode, vi isoleret mus i bugspytkirtlen Holme, kulturperler den isolerede islet celle kultur betingelse med kemikalier, og udnyttet den kemiske-induceret ROS generation som udlæsningen. Denne procedure er enkel og effektiv i at identificere ROS-inducerende kemikalier i isolerede Holmen; Det kan udvikles yderligere undersøgelse af de mekanismer af toksicitet, der er specifikke for bugspytkirtlen in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">10&#215;Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:283px;">GIBCO</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type 4</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLS-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysucrose/sodium diatrizoate solution&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#8217;,7&#8217;-dichlorofluorescein (DCFH-DA)</td>
			<td>Sigma</td>
			<td>D6883-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microplate reader&#160;</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptomycin</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>GIBCO</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">avertin</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat/Mouse Insulin ELISA Kit</td>
			<td>Millipore</td>
			<td>EZRMI-13K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorval</td>
			<td>Sorval RT7</td>
			<td>for 96-well plate centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td>Biotek</td>
			<td>Epoch 2</td>
			<td>for fluorescence reading</td>
		</tr>
	</tbody>
</table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Eksponering for visse miljømæssige kemikalier i mennesker og dyr er blevet fundet for at forårsage cellulære skader i bugspytkirtlen β-celler, hvilket vil føre til udviklingen af type 2 diabetes mellitus (T2DM). Selv om mekanismerne til den kemiske-induceret β celleskader var uklare og tilbøjelige til at være kompleks, er en tilbagevendende konstatering, at disse kemikalier fremkalde oxidativt stress fører til generation af overdreven reaktive ilt arter (ROS), fremkalde skader på β-cellen. For at identificere potentielle diabetogenic miljømæssige kemikalier, isoleret vi pancreas ø-celler fra C57BL/6 mus og kulturperler islet celler i 96-brønd celle kultur plader; derefter ø-celler blev doseret med kemikalier og ROS generation blev opdaget af 2', 7'-dichlorofluorescein (DCFH-DA) fluorescerende farvestof. Brug denne metode, vi fandt at bisphenol A (BPA), Benzo [a] pyren (BaP) og polychlorerede biphenyler (PCB), kunne fremkalde høje niveauer af ROS, tyder på, at de potentielt kan fremkalde skader i ø-celler. Denne metode bør være nyttige for screening diabetogenic fremmedstoffer. Derudover kan kulturperle ø-celler også tilpasses til in vitro- analyse af kemiske-induceret toksicitet i pancreas celler.

En Mikrograf af sunde isolerede Holmen er vist i figur 2, hvor Holme har en rund eller oval form med relativt ensartet størrelse (selvom størrelse ensartethed kan varierer fra stamme til stamme). Vi undersøgte næste funktionerne i bugspytkirtlen Holmen i en in vitro- analyse ved at isolere Holmen og stimulere insulinsekretion i kultur Holme. Figur 3 viser vores typiske analyse af GSIS analysen fra C57BL/6 mus isolere...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Her præsenterer vi en protokol for at isolere mus pancreas ø-celler for screening ROS induktion af fremmedstoffer for at identificere de potentielle diabetogenic xenobiotiske kemikalier.

En Murine pancreas Islet celle-baseret Screening for Diabetogenic miljømæssige kemikalier

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

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