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

Författarna har förklarat någon intressekonflikt.

Övergripande protokollet kräver 2 dagar, inklusive den första dagen av sepsis induktion och isolering av BAL vätska från djuret, och den andra dagen för exosomes isolering från musen BALF. Alla förfaranden har granskats och godkänts av institutionella djur vård och användning kommittén vid Atlanta VA medical center.
1. akut septisk Lung skada musmodell
<ol><li>Använd 6-8 veckor - gamla vildtyp C57BL/6J hanmöss (vikt 20-22 g) för djuret modellera och utför alla procedurer med ...

<ol>
	<li>Rubenfeld, G. D., Herridge, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+outcomes+of+acute+lung+injury.">Epidemiology and outcomes of acute lung injury.</a> Chest. 131, 554-562 (2007).</li><li>Baron, R. M., Choi, A. J., Owen, C. A., Choi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+manipulated+mouse+models+of+lung+disease:+potential+and+pitfalls.">Genetically manipulated mouse models of lung disease: potential and pitfalls.</a> American journal of physiology. Lung cellular and molecular physiology. , L485-L497 (2012).</li><li>Han, S., Mallampalli, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome:+from+mechanism+to+translation.">The acute respiratory distress syndrome: from mechanism to translation.</a> Journal of immunology. , 855-860 (1950).</li><li>Aeffner, F., Bolon, B., Davis, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Acute+Respiratory+Distress+Syndrome:+A+Review+of+Analytical+Approaches,+Pathologic+Features,+and+Common+Measurements.">Mouse Models of Acute Respiratory Distress Syndrome: A Review of Analytical Approaches, Pathologic Features, and Common Measurements.</a> Toxicologic pathology. 43, 1074-1092 (2015).</li><li>Matute-Bello, G., Frevert, C. W., Martin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+lung+injury.">Animal models of acute lung injury.</a> American journal of physiology. Lung cellular and molecular physiology. , L379-L399 (2008).</li><li>Terrasini, N., Lionetti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+Critical+Illness.">Exosomes in Critical Illness.</a> Critical care medicine. , (2017).</li><li>Fujita, Y., Kadota, T., Araya, J., Ochiya, T., Kuwano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles:+New+Players+in+Lung+Immunity.">Extracellular Vesicles: New Players in Lung Immunity.</a> American journal of respiratory cell and molecular biology. , (2017).</li><li>Kubo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles+in+Lung+Disease.">Extracellular Vesicles in Lung Disease.</a> Chest. 153, 210-216 (2018).</li><li>Krol, J., Loedige, I., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+widespread+regulation+of+microRNA+biogenesis,+function+and+decay.">The widespread regulation of microRNA biogenesis, function and decay.</a> Nature reviews. Genetics. 11, 597-610 (2010).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li><li>Fanini, F., Fabbri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CANCER-DERIVED+EXOSOMIC+microRNAs+SHAPE+THE+IMMUNE+SYSTEM+WITHIN+THE+TUMOR+MICROENVIRONMENT:+STATE+OF+THE+ART.">CANCER-DERIVED EXOSOMIC microRNAs SHAPE THE IMMUNE SYSTEM WITHIN THE TUMOR MICROENVIRONMENT: STATE OF THE ART.</a> Seminars in cell &amp; developmental biology. , (2016).</li><li>Agarwal, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental,+Systems,+and+Computational+Approaches+to+Understanding+the+MicroRNA-Mediated+Reparative+Potential+of+Cardiac+Progenitor+Cell-Derived+Exosomes+From+Pediatric+Patients.">Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients.</a> Circulation research. 120, 701-712 (2017).</li><li>Prattichizzo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microRNAs+and+endothelial+hyperglycaemic+memory:+a+therapeutic+opportunity?.">Extracellular microRNAs and endothelial hyperglycaemic memory: a therapeutic opportunity?.</a> Diabetes, obesity &amp; metabolism. 18, 855-867 (2016).</li><li>Huang-Doran, I., Zhang, C. Y., Vidal-Puig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles:+Novel+Mediators+of+Cell+Communication+In+Metabolic+Disease.">Extracellular Vesicles: Novel Mediators of Cell Communication In Metabolic Disease.</a> Trends in endocrinology and metabolism: TEM. 28, 3-18 (2017).</li><li>Khalyfa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Plasma+Extracellular+Microvesicle+MicroRNA+Cargo+and+Endothelial+Dysfunction+in+Children+with+Obstructive+Sleep+Apnea.">Circulating Plasma Extracellular Microvesicle MicroRNA Cargo and Endothelial Dysfunction in Children with Obstructive Sleep Apnea.</a> American journal of respiratory and critical care medicine. 194, 1116-1126 (2016).</li><li>Thomou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+circulating+miRNAs+regulate+gene+expression+in+other+tissues.">Adipose-derived circulating miRNAs regulate gene expression in other tissues.</a> Nature. 542, 450-455 (2017).</li><li>Gregson, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Exosomal+RNA+Profiles+in+Bronchoalveolar+Lavage+from+Lung+Transplants+with+Acute+Rejection.">Altered Exosomal RNA Profiles in Bronchoalveolar Lavage from Lung Transplants with Acute Rejection.</a> American journal of respiratory and critical care medicine. 192, 1490-1503 (2015).</li><li>Valadi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nature cell biology. 9, 654-659 (2007).</li><li>Doxaki, C., Kampranis, S. C., Eliopoulos, A. G., Spilianakis, C., Tsatsanis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+Regulation+of+miR-155+and+miR-146a+Genes+during+Induction+of+Endotoxin+Tolerance+in+Macrophages.">Coordinated Regulation of miR-155 and miR-146a Genes during Induction of Endotoxin Tolerance in Macrophages.</a> Journal of immunology. , 5750-5761 (2015).</li><li>Schulte, L. N., Westermann, A. J., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+and+functional+specialization+of+miR-146+and+miR-155+in+innate+immune+sensing.">Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing.</a> Nucleic acids research. 41, 542-553 (2013).</li><li>Alexander, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-delivered+microRNAs+modulate+the+inflammatory+response+to+endotoxin.">Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.</a> Nature communications. 6, 7321 (2015).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1+is+induced+in+tumor+associated+macrophages+by+cyclo-oxygenase+pathway+in+human+non-small+cell+lung+cancer.">TREM-1 is induced in tumor associated macrophages by cyclo-oxygenase pathway in human non-small cell lung cancer.</a> PloS one. 9, e94241 (2014).</li><li>Xu, R., Richards, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+In+Vitro+Co-Culture+Model+in+Anti-Cancer+Drug+Development+Cascade.">Development of In Vitro Co-Culture Model in Anti-Cancer Drug Development Cascade.</a> Combinatorial chemistry &amp; high throughput screening. 20, 451-457 (2017).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1-accentuated+lung+injury+via+miR-155+is+inhibited+by+LP17+nanomedicine.">TREM-1-accentuated lung injury via miR-155 is inhibited by LP17 nanomedicine.</a> American journal of physiology. Lung cellular and molecular physiology. , L426-L438 (2016).</li><li>Sadikot, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dose+dexamethasone+accentuates+nuclear+factor-kappa+b+activation+in+endotoxin-treated+mice.">High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice.</a> American journal of respiratory and critical care medicine. 164, 873-878 (2001).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+mediated+epigenetic+modulation+inhibits+TREM-1+expression+in+response+to+lipopolysaccharide.">Curcumin mediated epigenetic modulation inhibits TREM-1 expression in response to lipopolysaccharide.</a> The international journal of biochemistry &amp; cell biology. 44, 2032-2043 (2012).</li><li>Chauret, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorine+dioxide+inactivation+of+Cryptosporidium+parvum+oocysts+and+bacterial+spore+indicators.">Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators.</a> Applied and environmental microbiology. 67, 2993-3001 (2001).</li><li>Cizmar, P., Yuana, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+Characterization+of+Extracellular+Vesicles+by+Transmission+and+Cryo-Transmission+Electron+Microscopy.">Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy.</a> Methods in molecular biology. , 221-232 (2017).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-related+proteins+prolong+macrophage+survival+through+induction+of+Triggering+receptor+expressed+on+myeloid+cells-1.">HIV-related proteins prolong macrophage survival through induction of Triggering receptor expressed on myeloid cells-1.</a> Scientific reports. 7, 42028 (2017).</li><li>Jackson, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Mitochondrial+Transfer+in+Direct+Co-cultures+of+Human+Monocyte-derived+Macrophages+(MDM)+and+Mesenchymal+Stem+Cells+(MSC).">Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC).</a> Bio-protocol. 7, (2017).</li><li>Fujita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles:+New+Players+in+Lung+Immunity.">Extracellular Vesicles: New Players in Lung Immunity.</a> American journal of respiratory cell and molecular biology. 17, 474-484 (2017).</li><li>Gunasekaran, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-Derived+Exosomes+With+Lung+Self-Antigens+in+Human+Lung+Allograft+Rejection.">Donor-Derived Exosomes With Lung Self-Antigens in Human Lung Allograft Rejection.</a> American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 17, 474-484 (2017).</li><li>Kalra, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+evaluation+of+plasma+exosome+isolation+techniques+and+assessment+of+the+stability+of+exosomes+in+normal+human+blood+plasma.">Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.</a> Proteomics. 13, 3354-3364 (2013).</li><li>Baranyai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Exosomes+from+Blood+Plasma:+Qualitative+and+Quantitative+Comparison+of+Ultracentrifugation+and+Size+Exclusion+Chromatography+Methods.">Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.</a> PloS one. 10, e0145686 (2015).</li><li>Nordin, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafiltration+with+size-exclusion+liquid+chromatography+for+high+yield+isolation+of+extracellular+vesicles+preserving+intact+biophysical+and+functional+properties.">Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.</a> Nanomedicine : nanotechnology, biology, and medicine. 11, 879-883 (2015).</li></ol>

Mus-modeller av sjukdomar är vanligt förekommande att utvärdera den fysiologiska funktionen av specifika gener och minska kostnaden för experiment2. Den akut septisk lungskada som beskrivs här härmar inflammatorisk reaktion som ses hos människa med ARDS. Denna modell är relevant att undersöka molekylär patogenes, utvecklingen av biomarkörer och testa potentiella nya terapier5.
Samtidig kultur system är relevanta för in-vivo ...

ALI och ARDS är en livshotande form av andningssvikt med svår hypoxemi orsakad av icke-kardiogent lungödem som drabbar cirka 1 miljoner människor i världen årligen1. Etiologin av ARDS omfattar direkt skada på lungorna från infektioner eller aspiration och en mängd indirekta förolämpningar. Under det senaste decenniet har det skett en ökad förståelse för molekylär patogenes av ARDS, särskilda riktade behandlingar för ARDS är dock ännu vara utvecklat2,3.
Flera djurmodeller av Akut lungskada har utvecklats som ger en bro för att översätta experimentella behandlingar till humanstudier4,5. Ofta används modeller inkluderar lokal installation av oljesyra, bakterier, LPS och bleomycin. Andra metoder inkluderar ischemi-reperfusion, cecal ligering punktering, mekanisk ventilation-inducerad stretch skada, hyperoxia eller systemisk administrering av bakterier och LPS5. Dessa modeller ger ett användbart biologiska system för att testa kliniska hypoteser och för utvecklingen av potentiella behandlingar. För att simulera människors ARDS, bör djurmodeller återge inflammation och akut skada på epiteliala och endothelial celler med defekter i barriärfunktion i lungorna.
Exosomes är membranet blåsor med 20-200 nm i diameter, vars molekylära innehåll innehåller proteiner, DNA, RNA och lipider, och underlätta mellan mobilkommunikation i vävnad mikromiljö via molekylära sammansättning överföring. Exosomes utsöndras av flera typer av celler, endotelceller, epitelceller, glatta muskelceller och tumörceller, och finnas i mänskliga kroppsvätska. Studier visar att exosomes reglera överhörning mellan immunceller och stromaceller under smittsamma och sterila inflammatoriska sjukdomar, och deras onormala release verkar regleras genom olika naturliga och experimentell stimuli under fysiologiska och patologiska processer6. Sådan kommunikationsnätverk kan spela en viktig roll i patogenesen av lungsjukdomar och kan påverka patofysiologiska progression7,8. Som 18-22 nukleotid icke-kodande RNAs, MicroRNA finns i både vävnad och kroppsvätskor, plasma, serum och modulera mRNA uttryck posttranslationella nivå9,10.
Paketerade MicroRNA i exosomes påverkar differentiering och funktion av flera typer av celler, och alltför höga halter är associerade med en mängd olika sjukdomar, däribland cancer, lungsjukdomar, fetma, diabetes och hjärt-kärlsjukdom11, 12,13,14,15,16. Trätt i de mottagande cellerna och skytteltrafik av exosomal MicroRNA underlättar intercellulära kommunikationen modifiera hemostas mikromiljö17,18. Akut lungskada är en komplexa processer, som inbegriper flera celltyper med omfattande intercellulär kommunikation via exosomes8. miR-155 och miR-146a dela den gemensamma transkriptionell reglerande mekanismen och bidrar till den inflammatoriska reaktionen och immuntolerans19,20. Nyligen genomförda studier visar att både modulera inflammatoriska svar via exosomal MicroRNA skytteltrafik mellan immunceller21. Men de molekylära mekanismerna bakom immunmodulerande effekter av exosomal MicroRNA på alveolära svar på endotoxin förblir oklart, utan tvekan den potentiella kliniska relevansen och translationell implikation merit ytterligare undersökning.
Samtidig kultur modeller är att vara anställd att definiera samverkan mellan de specifika celltyper i komplexa miljön, såsom inflammation och cancer22,23. Dessa plattformar ger en alternativ strategi för att förhöra replikväxlning mellan celltyper särskilt för immun- och strukturella celler.
Intra-luftrör, aerosolized, intraperitoneal eller systemisk administrering av LPS används ofta för att framkalla experimentella lung skada24,25,26, och har visat för att inducera epitelial och endotel permeabilitet defekter. Här använder vi intraperitoneal LPS för att inducera en septisk modell av Akut lungskada hos möss. Inom 24 timmar efter intraperitoneal administrering av LPS induceras permeabilitet defekter i lungorna med rekrytering av inflammatoriska celler. Dessutom visar vi att exosomes från BAL innehåller miRNA-155 och miR-146a och exosomes från BAL vätska inducera pro-inflammatoriska cytokiner uttryck i mottagarens epitelceller, inklusive IL-6 och TNF-α. Dessa uppgifter är först att visa att exosomal MicroRNA utsöndras i BAL i denna modell av akut septisk lungskada.

<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 lungskada (ALI) och akut andnödssyndrom (ARDS) utgör en heterogen grupp av lungsjukdomar som fortsätter att ha en hög sjuklighet och dödlighet. Molekylär patogenes av ALI är att bättre definierade; på grund av sjukdomen komplexa har molekylära behandlingar dock ännu utvecklas. Här använder vi en lipopolysackarid (LPS) inducerade musmodell av akut septisk lungskada för att avgränsa rollen av exosomes i den inflammatoriska reaktionen. Använda denna modell, har vi kunnat visa att möss som utsätts för intraperitoneal LPS utsöndrar exosomes i Bronko-alveolar lavage (BAL) vätska i lungorna som paketeras med miRNA och cytokiner som reglerar inflammatoriska svar. Ytterligare med samtidig kultur modellsystem, visar vi att exosomes släppt från makrofager störa uttrycket av åtsittande föreningspunkt proteiner i bronkial epitelceller. Dessa resultat tyder på att 1) replikväxlning mellan medfödd immun- och strukturella celler genom den exosomal skytteltrafik bidrar till den inflammatoriska reaktionen och störningar av den strukturella hindren och 2) inriktning dessa MicroRNA föreskriva en roman plattform att behandla ALI och ARDS.

För att inducera septisk lungskada, behandlades möss med intraperitoneal LPS (15 mg/kg). Inom 24 h av LPS administration sågs neutrofil tillströmning i lungorna, som visas i figur 1A. Mus BAL vätska drogs, följt av isolering och rening av exosomes. Morfologi av BALF exosomes bekräftades av överföring elektronmikroskopi (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

Bronkoalveolär Lavage Exosomes i lipopolysackarid-inducerad septisk lungskada

Möss som utsätts för intraperitoneal LPS utsöndrar exosomes i Bronko-alveolar lavage (BAL) vätska som paketeras med microRNA. Samtidig kultur system med visar vi att exosomes släppt i BAL vätska störa uttrycket av åtsittande föreningspunkt proteiner i bronkial epitelceller och öka uttrycket av pro-inflammatoriska cytokiner som framhäver lungskada.

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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