Name: bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury
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Den samlede protokollen kræver 2 dage, herunder den første dag af sepsis induktion og isolation af BAL væske fra dyret, og den anden dag for exosomes isolation fra mus BALF. Alle procedurer er gennemgået og godkendt af institutionelle Animal Care og brug Udvalget på Atlanta VA medical center.
1. Mouse akut septisk lunge skade Model
<ol><li>Brug 6-8 uger - gamle mandlige vildtype C57BL/6J mus (vægt 20-22 g) for dyret model, og udføre alle procedurer ved hjælp af aseptisk teknikker og ...

<ol>
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Musemodeller af sygdomme er almindeligt anvendt til at evaluere den fysiologiske funktion af specifikke gener og reducere omkostningerne ved eksperimenter2. Den akutte septiktanke lunge skade beskrevet her efterligner inflammatorisk reaktion ses hos mennesker med ARDS. Denne model er relevante at undersøge den molekylære patogenese, udvikling af biomarkører og teste potentielle nye behandlingsformer5.
Fælles kultur systemer er relevante for <...

ALI og ARDS er de livstruende former for respiratorisk svigt med svær hypoxæmi forårsaget af ikke-kardiogent lungeødem, som påvirker omkring 1 million mennesker verden over hvert år1. Ætiologien af ARDS omfatter direkte skade på lungerne fra infektioner eller aspiration og en række indirekte fornærmelser. I det seneste årti har der været en øget forståelse for de molekylære patogenese af ARDS, specifikke målrettede behandlinger for ARDS er dog endnu at være udviklet2,3.
Flere dyremodeller for akut lunge skade er blevet udviklet som giver en bro til at oversætte eksperimentelle behandlinger til humane undersøgelser4,5. Almindeligt anvendte modeller omfatter lokale installation af oliesyre, bakterier, LP'ER og bleomycin. Andre metoder omfatter iskæmi-reperfusion, cecal ligatur punktering, mekanisk ventilation-induceret stretch skade, Iltforgiftning eller systemisk administration af bakterier og LP'ER5. Disse modeller giver en nyttig biologiske system for at teste kliniske hypoteser og for udvikling af mulige behandlinger. For at simulere menneskelig ARDS, bør dyremodeller reproducere inflammation og akut skade de epitel og endothelial celler med defekter i barriere funktion i lungerne.
Exosomes er membran vesikler med 20-200 nm i diameter, hvis molekylære indhold indeholder proteiner, DNA, RNA og lipider, og lette inter cellular meddelelser i væv mikromiljø via molekylære sammensætning overførsel. Exosomes udskilles af flere typer af celler, såsom endotelceller, epitelceller, glatte muskelceller og tumorceller, og findes i menneskelige legeme væske. Undersøgelser tyder på, at exosomes regulere cross-talk mellem immunceller og stromale celler under smitsom og sterile inflammatoriske sygdomme, og deres unormale udgivelse ser ud til at være underlagt forskellige naturlige og eksperimenterende stimuli under fysiologiske og patologiske processer6. Sådanne kommunikationsnet kan spille en vigtig rolle i patogenesen af lungesygdomme og kan påvirke patofysiologiske progression7,8. Som 18-22 nukleotid ikke-kodende RNA'er, miRNAs findes i både væv og legemsvæsker, plasma, sera og modulere mRNA udtryk på posttranslationel niveau9,10.
Emballerede miRNAs i exosomes påvirke differentiering og funktion af flere typer af celler, og overdreven niveauer er forbundet med en lang række sygdomme, herunder kræft, lungesygdomme, fedme, diabetes og hjerte-kar-sygdom11, 12,13,14,15,16. Indrejse i de modtagende celler og shuttling af exosomal miRNAs lette intercellulær kommunikation modificerer hæmostase mikromiljø17,18. Akut lunge-skade er en komplekse processer, der involverer flere celletyper med omfattende intercellulær kommunikation via exosomes8. miR-155 og miR-146 deler den fælles transcriptional regulerende mekanisme og bidrage til det inflammatoriske respons og immuntolerance19,20. Nylige undersøgelser tyder på at både modulere inflammatoriske respons via exosomal miRNAs shuttling mellem immunceller21. Men de molekylære mekanismer bag modulerende virkningerne af exosomal miRNAs på alveolær svar på endotoxin forbliver uklart, utvivlsomt potentielle klinisk relevans og translationel Implikationen merit yderligere undersøgelse.
Fælles kultur modeller er ansat til at definere interaktionen mellem de specifikke celletyper i det komplekse miljø, såsom betændelse og cancer22,23. Disse platforme giver en alternativ strategi for at afhøre krydstale mellem celletyper især for immun og strukturelle celler.
Intra-luftrør, aerosolmaterialer, intraperitoneal eller systemisk administration af LP'ER er almindeligt anvendt til at fremkalde eksperimentelle lunge skade24,25,26, og har vist sig for at fremkalde epitel og endotel permeabilitet defekter. Her bruger vi intraperitoneal LP'ER til at fremkalde en septisk model af akut lunge skade i mus. Inden for 24 timer af intraperitoneal indgift af LP'ER, er permeabilitet defekter induceret i lungerne med rekruttering af inflammatoriske celler. Vi viser yderligere, at exosomes fra BAL indeholder miRNA-155 og miR-146, og exosomes fra BAL væske fremkalde pro-inflammatoriske cytokiner udtryk i modtagerens epitelceller, herunder IL-6 og TNF-α. Disse data er de første til at vise at exosomal miRNAs udskilles i BAL i denne model af akut septisk lunge skade.

<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

Akut lunge skade (ALI) og Akut respiratorisk distress syndrom (ARDS) repræsenterer en heterogen gruppe af lungesygdomme, som fortsat har en høj sygelighed og dødelighed. Den molekylære patogenese af ALI bliver bedre defineret; men på grund af den komplekse karakter af sygdommen molekylære behandlingsformer har endnu skal udvikles. Her bruger vi en LPS (LPS) induceret musemodel af akut septisk lunge skade for at afgrænse rollen som exosomes i det inflammatoriske respons. Bruger denne model, har vi kunnet vise, at mus, der er udsat for intraperitoneal LP'ER udskiller exosomes i bronchopneumoni-alveolære lavage (BAL) væske fra lungerne, som er pakket med miRNA og cytokiner, der regulerer inflammatorisk respons. Yderligere ved hjælp af en fælles kultur modelsystem, viser vi, at exosomes frigivet fra makrofager forstyrrer udtryk for tight junction proteiner i bronchiale epitel celler. Disse resultater tyder på, at 1) krydstale mellem medfødte immun og strukturelle celler gennem exosomal shuttling bidrage til det inflammatoriske respons og afbrydelse af den strukturelle barriere og 2) rettet mod disse miRNAs kan give en ny platform til at behandle ALI og ARDS.

For at fremkalde septisk lunge skade, blev mus behandlet med intraperitoneal LP'ER (15 mg/kg). Inden for 24 timer af LP'ER administration, blev neutrophilic tilstrømningen set i lungerne, som vist i figur 1A. Musen BAL væske var udarbejdet, efterfulgt af isolering og oprensning af exosomes. Morfologi af BALF exosomes blev bekræftet af elektron transmission mikroskopi (figur 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 i LPS-induceret septisk lunge skade

Mus udsættes for intraperitoneal LP'ER udskiller exosomes i bronchopneumoni-alveolære lavage (BAL) væske, som er pakket med miRNAs. Ved hjælp af en fælles kultur system, viser vi, at exosomes udgivet i BAL væske forstyrre udtryk for tight junction proteiner i bronchiale epitel celler og øge udtryk af pro-inflammatoriske cytokiner, at fremhæver lunge skade.

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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