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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Den generelle protokollen krever 2 dager, inkludert den første dagen av sepsis induksjon og isolering av BAL væske fra dyret, og den andre dagen for exosomes isolasjon fra musa BALF. Alle prosedyrer har blitt vurdert og godkjent av institusjonelle Animal Care og bruk komiteen ved Atlanta VA medical center.
1. mus akutt septisk lungen skade modell
<ol><li>Bruker 6-8 uker - gammel mannlig vill-type C57BL/6J mus (vekt 20-22 g) for dyret modell, og utfører alle prosedyrer som bruker steril te...

<ol>
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Musen modeller av sykdommer er vanlig å evaluere bestemte gener fysiologiske funksjon og redusere kostnadene for eksperimentering2. Akutt septisk lunge skaden beskrevet her etterligner betennelsesreaksjon sett i mennesker med ARDS. Denne modellen er relevant å undersøke molekylær patogenesen, utvikling av biomarkers og teste potensielle nye terapier5.
Co kultur systemer er relevante for i vivo studier i sammenheng med undersøker pot...

ALI og ARDS er livstruende former av respirasjonssvikt med alvorlig hypoksemi forårsaket av ikke-kardiogent lungeødem som påvirker ca 1 million mennesker over hele verden årlig1. Etiologien for ARDS omfatter direkte skade lungene fra infeksjoner eller aspirasjon og en rekke indirekte fornærmelser. Det siste tiåret har det vært en økt forståelse av molekylære patogenesen av ARDS, men bestemt målrettet behandlinger for ARDS er ennå å bli utviklet2,3.
Flere dyr modeller av akutt lungen skade har blitt utviklet som gir en bro for å oversette eksperimentell terapi til menneskelige studier4,5. Brukte modeller inkluderer lokal installasjon av oljesyre, bakterier, LP-plater og bleomycin. Andre metoder inkluderer ischemia-reperfusion, cecal ligation punktering, mekanisk ventilasjon-indusert strekk skade, hyperoxia eller systemisk administrasjon av bakterier og LPS5. Disse modellene gir en nyttig biologiske system for å teste klinisk hypoteser og for utvikling av potensielle terapier. For å simulere menneskelige ARDS, bør dyremodeller reprodusere betennelse og akutt skade epitel og endothelial cellene med defekter i barriere-funksjonen i lungene.
Exosomes er membran blemmer med 20-200 nm i diameter, som molekylær innhold inneholder proteiner, DNA, RNA og lipider, og lette Inter cellular kommunikasjon i vev microenvironment Met molekylær komposisjon. Exosomes utskilles av flere typer celler, for eksempel endotelceller, epitelceller, glatt muskelceller og kreftceller, og finnes i menneskelige kroppen væske. Studier indikerer at exosomes regulere kryss-snakk mellom immunceller og stromal celler under smittsomme og sterile inflammatoriske sykdommer, og deres unormale utgivelse synes å være regulert av ulike naturlige og eksperimentelle stimuli under fysiologiske og patologisk prosesser6. Slike kommunikasjonsnettverk kan spille en viktig rolle i patogenesen av lungesykdommer og kan påvirke patofysiologiske progresjon7,8. Som 18-22 nukleotid ikke-koding RNAs, miRNAs finnes i både vev og kroppsvæsker, plasma, sera, og modulere mRNA uttrykk på post-translasjonell nivå9,10.
Pakket miRNAs i exosomes påvirke differensiering og funksjon av flere typer celler, og overdreven nivåer er knyttet til en rekke sykdommer, inkludert kreft, lungesykdommer, fedme, diabetes og hjerte-og karsykdommer11, 12,13,14,15,16. Mottaker cellene og skytteltrafikk av exosomal miRNAs lette intercellulære kommunikasjon endre hemostasen microenvironment17,18. Akutt lunge-skade er en komplekse prosesser, som involverer flere celletyper med omfattende intercellulære kommunikasjon gjennom exosomes8. miR-155 og miR-146a dele felles transcriptional regulatoriske mekanismen og bidra til inflammatorisk respons og immun toleranse19,20. Nyere studier viser at begge modulere inflammatorisk respons via exosomal miRNAs skytteltrafikk mellom immunceller21. Men molekylære mekanismer underliggende modulatory effekter av exosomal miRNAs på alveolar svar på endotoxin fortsatt uklare, utvilsomt potensielle klinisk betydning og translasjonsforskning implikasjon fortjener videre etterforskning.
Co kultur modeller brukes til å definere samspillet av de spesifikke celletyper i komplekse miljøet, for eksempel betennelse og kreft22,23. Disse plattformene gir en alternativ strategi for å avhøre kryss-snakk mellom celletyper spesielt for immun og strukturelle celler.
Intra-tracheal, aerosolized, intraperitoneal eller systemisk administrasjon av LPS brukes å indusere eksperimentelle lungen skade24,25,26, og har vist for å indusere epitel og endothelial permeabilitet defekter. Her bruker vi intraperitoneal LPS for å indusere en septisk modell av akutt lungen skade i mus. Innen 24 timer for intraperitoneal administrasjon av LPS, er permeabilitet defekter indusert i lungene med rekruttering av inflammatoriske celler. Videre, vi viser at exosomes fra BAL inneholder miRNA-155 og miR-146a, og exosomes fra BAL væske induserer pro-inflammatoriske cytokiner uttrykk i mottakerens epitelceller, inkludert IL-6 og TNF-α. Disse dataene er først til å vise at exosomal miRNAs utskilles i BAL i denne modellen av akutt septisk lungen 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

Akutt lungen skade (ALI) og akutt respiratory distress syndrom (ARDS) representerer en heterogen gruppe av lungesykdommer som fortsetter å ha en høy sykelighet og dødelighet. Molekylær patogenesen av ALI blir bedre definert. men på grunn av den komplekse naturen av sykdommen har Molekylær terapi ennå å bli utviklet. Her bruker vi en lipopolysakkarid (LPS) indusert musemodell av akutt septisk lungen skade for å avgrense rollen exosomes i betennelsesreaksjon. Bruker denne modellen, kunne vi vise at mus er utsatt for intraperitoneal LPS skiller exosomes i Broncho-alveolar lavage (BAL) fluid fra lungen som er pakket med miRNA og cytokiner som regulerer betennelsesreaksjon. Videre bruker en co kultur modellsystem, viser vi at exosomes fra makrofager forstyrre uttrykk for tett krysset proteiner i bronkial epitelceller. Disse resultatene tyder på at 1) kryss-snakk mellom medfødte immunsystemet og strukturelle cellene til det exosomal skytteltrafikk bidrar til inflammatorisk respons og forstyrrelse av strukturelle barrieren og 2) rettet mot disse miRNAs kan gi en ny plattform for å behandle ALI og ARDS.

For å indusere septisk lungen skade, ble mus behandlet med intraperitoneal plater (15 mg/kg). Innen 24 timer av LPS administrasjon, ble neutrophilic tilstrømningen sett i lungene, som vist i figur 1A. Musen BAL væske ble trukket, etterfulgt av isolasjon og rensing av exosomes. Morfologi av BALF exosomes ble bekreftet av elektronmikroskop overføring (figur 1B).
Uttry...

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 lipopolysakkarid-indusert septisk lungen skade

Mus utsatt for intraperitoneal LPS skiller exosomes i Broncho-alveolar lavage (BAL) væske som er pakket med miRNAs. Bruker en co kultur, viser vi at exosomes utgitt i BAL væske forstyrre uttrykk for tett krysset proteiner i bronkial epitelceller og øke uttrykk for pro-inflammatoriske cytokiner som fremhever lungen 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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