Name: bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury
Uploaded: 2018-05-21T15:00:00
Description: Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

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Het totale protocol vereist 2 dagen, met inbegrip van de eerste dag van sepsis inductie en isolatie van BAL vloeistof uit het dier, en de tweede dag voor exosomes isolatie van de muis BALF. Alle procedures zijn herzien en goedgekeurd door het institutionele Animal Care en gebruik Comité aan Atlanta VA medical center.
1. Acute septische Lung Injury muismodel
<ol><li>Gebruik 6-8 weken - oude mannelijke wild-type C57BL/6J muizen (gewicht 20-22 g) voor het dier model, en uitvoeren van alle proc...

<ol>
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Muismodellen van ziekten worden vaak gebruikt om te evalueren van de fysiologische functie van specifieke genen en om de kosten van experimenten2. De acute septische Long schade beschreven hier bootst ontstekingsreactie gezien bij mensen met ARDS. Dit model is relevante onderzoeken de Moleculaire Pathogenese, ontwikkeling van biomarkers en het testen van mogelijke nieuwe therapieën5.
Co cultuur systemen zijn relevant voor in vivo onderz...

ALI en ARDS zijn de levensbedreigende vormen van respiratoire insufficiëntie met ernstige hypoxemia veroorzaakt door niet-cardiogeen longoedeem die ongeveer 1 miljoen mensen wereldwijd treft jaarlijks1. De etiologie van ARDS omvat directe schade aan de longen van infecties of aspiratie en een scala aan indirecte beledigingen. De afgelopen tien jaar is er een toegenomen begrip van de moleculaire pathogenese van ARDS, specifieke gerichte behandelingen voor ARDS zijn echter nog worden ontwikkeld2,3.
Verschillende dierlijke modellen van acute long letsel hebben ontwikkeld die voorzien in een brug vertalen van experimentele therapieën op menselijke studies4,5. Gebruikte modellen omvat lokale installatie van oliezuur, bacteriën, LPS en bleomycine. Andere benaderingen omvatten ischemie-reperfusie, cecal afbinding punctie, mechanische ventilatie-geïnduceerde stretch letsel, hyperoxia of systemische toediening van bacteriën en LPS5. Deze modellen bieden een nuttige biologisch systeem om te testen van klinische hypothesen en voor de ontwikkeling van mogelijke therapieën. Als u wilt nabootsen menselijke ARDS, moeten dierlijke modellen reproduceren ontsteking en acute schade aan de epitheliale en endotheliale cellen met afwijkingen in de functie van de barrière in de longen.
Exosomes zijn membraan blaasjes met 20-200 nm in diameter, waarvan de moleculaire inhoud bevat eiwitten, DNA, RNA en lipiden, en vergemakkelijken van Inter cellulaire communicatie in weefsel communicatie via moleculaire samenstelling overdracht. Exosomes worden uitgescheiden door meerdere soorten cellen, zoals de endotheliale cellen, epitheliaale cellen, zachte spiercellen en tumorcellen, en bestaan in menselijke lichaamsvloeistof. Studies tonen aan dat exosomes cross-talk tussen immuuncellen en stromale cellen tijdens de bestrijding van infectiekiemen en steriele ontstekingsziekten reguleren, en hun abnormale vrijlating lijkt te worden geregeld door verschillende natuurlijke en experimentele prikkels tijdens fysiologische en ziekteprocessen6. Dergelijke communicatie-netwerk kan een belangrijke rol spelen in de pathogenese van longziekten en pathofysiologische progressie7,8kan beïnvloeden. Zo 18-22 nucleotide niet-coderende RNAs, miRNAs bestaan in zowel weefsels en lichaamsvloeistoffen, plasma, sera en moduleren van mRNA uitdrukking bij posttranslationele niveau9,10.
Verpakte miRNAs in exosomes differentiatie en functie van meerdere soorten cellen beïnvloeden, en buitensporige niveaus worden geassocieerd met een verscheidenheid van ziekten, waaronder kanker, longziekten, obesitas, diabetes en hart-en vaatziekten11, 12,13,14,15,16. Inwerkingtreding van de ontvangende cellen en pendelen van exosomal miRNAs vergemakkelijken intercellulaire communicatie wijzigen de hemostase communicatie17,18. Acute Long-blessure is een complexe processen, waarbij meerdere celtypen met uitgebreide intercellulaire communicatie via exosomes8. miR-155 en miR-146a delen van de gemeenschappelijke transcriptionele reguleringsmechanisme en bijdragen aan de ontstekingsreactie en de immuun tolerantie19,20. Recente studies tonen aan dat zowel ontstekingsreactie moduleren via exosomal miRNAs pendelen tussen immuuncellen21. Echter, de moleculaire mechanismen die ten grondslag liggen aan f effecten van exosomal miRNAs op de alveolaire reactie op endotoxine onduidelijk blijven, ongetwijfeld de potentiële klinische relevantie en translationeel implicatie verdienste verder onderzoek.
Co cultuur modellen gebruikt om te definiëren van de wisselwerking tussen de specifieke celtypes in de complexe omgeving, zoals de ontsteking en kanker22,23. Deze platforms bieden een alternatieve strategie te ondervragen cross talk tussen celtypen, met name voor immuun en structurele cellen.
Intra-tracheale, aërosol, intraperitoneaal of systemische toediening van LPS worden veel gebruikt voor het opwekken van experimentele long letsel24,25,26, en voor het opwekken van epitheliale en endotheel permeabiliteit is gebleken defecten. We gebruiken hier intraperitoneaal LPS ertoe een septische model van acute lung injury in muizen. Binnen de 24u intraperitoneale toediening van LPS, permeabiliteit defecten veroorzaakt in de longen met de aanwerving van ontstekingscellen. Verder laten we zien dat de exosomes van de BAL miRNA-155 en miR-146a bevatten, en exosomes aan de BAL vloeistof opwekken van pro-ontsteking cytokinen expressie in ontvangende epitheliale cellen, met inbegrip van IL-6 en TNF-α. Deze gegevens zijn de eerste om te laten zien dat exosomal miRNAs bal in dit model van acute septische long letsel worden uitgescheiden.

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Acute long letsel (ALI) en acute respiratory distress syndrome (ARDS) vormen een heterogene groep van longziekten die nog steeds een hoge morbiditeit en mortaliteit. De moleculaire pathogenese van ALI wordt beter gedefinieerd; echter, vanwege de complexe aard van de ziekte moleculaire therapieën moeten nog worden ontwikkeld. Hier gebruiken we een lipopolysaccharide (LPS) geïnduceerde muismodel van acute septische long letsel af te bakenen de rol van exosomes in de inflammatoire respons. Met behulp van dit model, hebben wij laten zien dat muizen die zijn blootgesteld aan intraperitoneaal LPS afscheiden exosomes in Broncho-alveolaire lavage (BAL) vloeistof uit de longen die zijn ingepakt met de miRNA en cytokinen tot regeling van de ontstekingsreactie. Verder gebruikt een modelsysteem co cultuur, laten we zien dat exosomes verlost van macrofagen expressie van strakke junction eiwitten in bronchiale epitheliale cellen verstoren. Deze resultaten suggereren dat 1) cross talk tussen aangeboren immuun en structurele cellen via de exosomal pendelen bijdraagt aan het inflammatoire respons en verstoring van de structurele barrière en 2) gericht op deze miRNAs een nieuw platform bieden kan te behandelen ALI en ARDS.

Om te induceren septische long letsel, werden muizen behandeld met intraperitoneaal LPS (15 mg/kg). Binnen de 24u van LPS administratie, werd neutrofiele toestroom gezien in de longen, zoals weergegeven in figuur 1A. Muis BAL vloeistof werd getrokken, gevolgd door isolatie en zuivering van exosomes. Morfologie van BALF exosomes werd bevestigd door Transmissie Electronenmicroscopie (figuur 1B).
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Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Bronchoalveolar Lavage Exosomes Lipopolysaccharide-geïnduceerde septische long letsel

Muizen blootgesteld aan intraperitoneaal LPS afscheiden exosomes in Broncho-alveolaire lavage (BAL) vloeistof die zijn ingepakt met miRNAs. Met behulp van een systeem co cultuur tonen we dat exosomes vrijgegeven in de BAL vloeistof expressie van strakke junction eiwitten in bronchiale epitheliale cellen verstoren en verhogen van expressie van pro-ontsteking cytokinen die long letsel accentueren.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

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