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 ...

The overall goal of this experiment is to delineate the role of exosomes in the inflammatory responses in an LPS induced, acute septic lung injury mouse model. So this method can help answer key questions related to cell cross talk in acute lung injury and acute respiratory distress syndrome. Especially intercellular communication within the alveolar-micro environment via exosome shuttling.The main advantage of this technique is that it allows the isolation and characterization of highly purified bronchoalveolar lavage exosomes derived from endotoxin treated septic mice with acute lung injury. Demonstrating the procedure is Dr.Zhihong Yuan and Dr.Chetna Bedi. To begin, load a one milliliter syringe equipped with a 27 gauge needle with 0.1 milliliters of LPS and PBS per mouse and manually restrain the first six to eight week old male C57 black 6J mouse.Deliver the LPS intra-peritoneally and return the animal to its cage. 24 hours later, make a five millimeter incision in the center of the neck of the first animal and use blunt forceps to gently separate the muscles to expose the trachea. Next, gently insert a 27 gauge needle attached to a sterile 10 milliliter syringe into the trachea and inject one milliliter of sterile PBS.Then immediately replace the 10 milliliter syringe with a three milliliter syringe equipped with a new 27 gauge needle. Aspirate the bronchoalveolar lavage fluid or BOLF and add the BOLF to an appropriately labeled 15 milliliter conical tube on ice. To isolate the exosomes, centrifuge the BOLF samples and transfer the supernatants to new 15 milliliter tubes.Centrifuge the samples again and transfer the supernatants to ultra-centrifuge tubes to remove the larger, cellular particles. Transfer the supernatants to new conical tubes and load the samples into 0.2 micron syringe filters to remove any remaining larger particles. Add the filtered samples to new ultra-centrifuge tubes to pellet the exosomes.Carefully discard the supernatants and re-suspend the pellets in 100 microliters of PBS per tube. Then transfer the exosome samples into individual 1.5 milliliter tubes for their minus 80 degree Celsius storage. To purify the exosomes, add 0.4 microliters of the appropriate exosome dye to one tube containing 100 microliters of suspension solution per sample and quickly add one thawed exosome sample to each tube of dye.Thoroughly mix the exosomes with the dye and incubate the samples at room temperature for five minutes, protected from light. At the end of the incubation, place one exosome spin column per sample into a 1.5 milliliter illusion tube and transfer the exosome solutions to the center of each spin column. Then centrifuge the samples, discard the columns, and store the alluded samples at minus 20 degrees Celsius.To measure exosome uptake by recipient cells, first seed one times 10 to the fourth mouse lung epithelial 12 cells in 0.5 milliliters of medium into each well of an eight well chamber slide for a 16 hour culture at 37 degrees Celsius in 5%carbon dioxide. After thawing, add 10 microliters of the purified fluorescent labeled exosomes to each well and return the cell cultures to the incubator for one hour. At the end of the incubation, wash the cells with PBS and fix each co-culture with 1%paraformaldehyde for 10 minutes at room temperature.Remove the medium and then mount the slide with medium supplemented with DAPI and image the samples by fluorescence microscopy at a 40 times magnification. To verify exosome interaction with recipient cells, seed two times 10 to the fifth mouse lung epithelial 12 cells in two milliliters of medium per well in a 12 well culture plate containing the appropriate culture medium for a two hour incubation at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, add 20 micrograms of exosomes to three wells of epithelial cells per experimental group and return the cells to the incubator.After 24 hours, remove the medium, wash the cells with PBS, and add 600 microliters of lysis buffer to each well with gentle shaking. Then transfer the entire contents of each well into individual 1.5 milliliter tubes and analyze the samples according to the standard real time PCR protocols. Within 24 hours of LPS administration, neutrophilic influx is observed in the lungs of LPS treated animals.After their isolation and purification as just demonstrated, the presence of exosomes in the BOLF from septic lung injury animals can be confirmed by transmission electron microscopy. The expression of select pro-inflammatory micro RNA is significantly increased in bronchoalveolar fluid exosomes from LPS treated animals compared to controlled PBS treated mice. LPS treated exosomes are taken up by mouse bronchiole epithelial cells as evidenced by the co-localization of fluorescent dye labeled exosomes with DAPI labeled cells after one hour of co-culture.Pro-inflammatory mediator expression is significantly increased in epithelial cells co-cultured with exosomes from LPS treated animals compared to co-culture with exosomes from control PBS treated mice. Further, cells treated with exosomes from LPS treated mice demonstrate a significantly attenuated expression of zonula occludens one compared to controls indicating a less intact cell integrity after LPS treated exosome co-culture. The intra-peritoneal injection of LPS, the bronchoalveolar lavage extraction, and exosome delegation from the bronchoalveolar lavage all the steps can be completed in a relatively short amount of time.Following this procedure, other methods like exosome and epithelial cell co-culture can be performed to answer additional questions about the cross talk between innate and structural cells in the lung alveolar micro-environment. Cross talk between innate immune and structural cells via exosome shuttling contributes to the inflammatory response and to structural barrier disruption suggesting exosomal micro-RNA targeting may provide a normal platform for treating acute lung injury and acute respiratory distress syndrome. After watching this video you should have a good understanding of how to induce septic lung injury in mice, isolate bronchoalveolar lavage fluid from mice, isolate exosomes from the lavage fluid, and use a co-culture model for interrogating intracellular cross talk.Don't forget that working with LPS can be hazardous and that precautions such as wearing the appropriate personal protective equipment should always be taken while performing this procedure.

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