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

Os autores declararam que não há conflito de interesses.

O protocolo global requer 2 dias, incluindo o primeiro dia da indução de sepse e isolamento do fluido BAL de animais e o segundo dia para isolamento de exosomes do mouse BALF. Todos os procedimentos foram revistos e aprovados pelo Comitê de uso no centro médico do VA de Atlanta e institucional Cuidado Animal.
1. modelo de lesão pulmonar aguda séptico Mouse
<ol><li>Use 6-8 semanas - velho selvagem-tipo C57BL/6J camundongos machos (20 a 22 g de peso) para o animal modelo e executar todos...

<ol>
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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. 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Modelos de mouse de doenças são comumente usados para avaliar a função fisiológica de genes específicos e para reduzir o custo de experimentação2. A lesão pulmonar aguda séptico descrita aqui imita a resposta inflamatória nos seres humanos com SDRA. Este modelo é relevante para investigar a patogénese molecular, desenvolvimento de biomarcadores e testar potenciais novas terapias5.
Sistemas de co-cultura são relevantes para estudos <...

ALI e Sara é as formas de vida-ameaçando de insuficiência respiratória com hipoxemia grave causada por edema pulmonar não-cardiogênico, que afeta cerca de 1 milhão pessoas no mundo anualmente1. A etiologia da SDRA inclui lesão direta para os pulmões de infecções ou aspiração e uma variedade de insultos indiretas. Na última década, tem havido uma maior compreensão da patogênese molecular da SDRA, no entanto, tratamentos alvo específicos para SDRA estão ainda a ser desenvolvido,2,3.
Foram desenvolvidos vários modelos animais de lesão pulmonar aguda que fornecem uma ponte para traduzir terapias experimentais para estudos humanos4,5. Modelos comumente usados incluem a instalação local do ácido oleico, bactérias, LPS e bleomicina. Outras abordagens incluem isquémia-reperfusão, punção cecal ligadura, lesão de estiramento induzida pela ventilação mecânica, hiperóxia ou administração sistémica de bactérias e LPS-5. Esses modelos fornecem um sistema biológico útil para testar hipóteses clínicas e para o desenvolvimento de terapias potenciais. Para simular a SDRA humana, modelos animais devem reproduzir inflamação e lesão aguda para as células epiteliais e endoteliais com defeitos na função de barreira nos pulmões.
Exosomes são vesículas de membrana com 20-200 nm de diâmetro, cujo conteúdo molecular contém proteínas, DNA, RNA e lipídios, e facilitar a comunicação intercelular no microambiente tecido via transferência de composição molecular. Exosomes são secretadas por vários tipos de células, como células endoteliais, células epiteliais, células musculares lisas e células tumorais e existem em fluidos do corpo humano. Estudos indicam que a exosomes regular conversas cruzadas entre células do estroma e células do sistema imunológico durante doenças inflamatórias, infecciosas e estéril, e sua liberação anormal parece ser regulada por vários estímulos naturais e experimentais durante fisiológica e processos patológicos6. Essa rede de comunicação pode desempenhar um importante papel na patogênese de doenças pulmonares e pode afetar a progressão fisiopatológicos7,8. 18-22 como nucleotídeos RNAs não-codificantes, miRNAs existem em ambos os tecidos e fluidos corporais, plasma, soro e modulam a expressão de RNAm no nível pós-traducional9,10.
MiRNAs embalados em exosomes influenciam a diferenciação e função dos vários tipos de células, e níveis excessivos são associados com uma variedade de doenças, incluindo câncer, doenças pulmonares, obesidade, diabetes e doença cardiovascular11, 12,13,14,15,16. Entrada em células da destinatários e vaivém de miRNAs exosomal facilitar a comunicação intercelular, modificando a hemostasia do microambiente17,18. Lesão pulmonar aguda é um processos complexos, envolvendo vários tipos de células com extensa comunicação intercelular através de exosomes8. miR-155 e miR-146 compartilham o mecanismo regulador transcricional comum e contribuam para a resposta inflamatória e a tolerância imunológica19,20. Estudos recentes indicam que ambos modulam a resposta inflamatória via exosomal miRNAs vaivém entre células do sistema imunológico21. No entanto, os mecanismos moleculares subjacentes moduladora efeitos de miRNAs exosomal na resposta alveolar a endotoxina permanecem pouco claras, sem dúvida, o potencial relevância clínica e translacional implicação mérito mais investigações.
Modelos de co-cultura estão sendo empregados para definir a interação dos tipos de célula específica no complexo ambiente, tais como inflamação e câncer22,23. Estas plataformas fornecem uma estratégia alternativa para interrogar conversas cruzadas entre tipos de células, particularmente por células imunes e estruturais.
Intratraqueal, aerossol, intraperitoneal ou sistêmica administração de LPS são amplamente utilizados para induzir a lesão de pulmão experimental a24,25,26e mostraram para induzir permeabilidade endotelial e epitelial defeitos de fabricação. Aqui usamos LPS intraperitoneal para induzir um fossa séptico modelo de lesão pulmonar aguda em camundongos. Dentro de 24 horas da administração intraperitoneal de LPS, permeabilidade defeitos são induzidos nos pulmões com recrutamento de células inflamatórias. Além disso, mostramos que exosomes de BAL contêm miRNA-155 e miR-146, e exosomes de fluido BAL induzir a expressão de citocinas pró-inflamatórias em pilhas epithelial do destinatários, incluindo IL-6 e TNF-α. Estes dados são os primeiros a mostrar que os miRNAs exosomal são secretadas no BAL neste modelo de lesão pulmonar aguda séptico.

<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

Lesão pulmonar aguda (LPA) e síndrome da angústia respiratória (Sara) representam um grupo heterogêneo de doenças pulmonares, que continua a ter uma alta taxa de morbidade e mortalidade. A patogénese molecular de ALI está sendo melhor definido; no entanto, devido à natureza complexa da doença terapias moleculares ainda precisam ser desenvolvido. Aqui nós usamos um modelo de rato induzida de lipopolissacarídeo (LPS) de lesão pulmonar aguda séptico para delinear o papel do exosomes na resposta inflamatória. Usando este modelo, fomos capazes de mostrar que os ratos expostos a LPS intraperitoneal secretam exosomes no líquido de lavagem (BAL) bronco-alveolar dos pulmões que são embalados com miRNA e citocinas que regulam a resposta inflamatória. Ainda mais usando um sistema de modelo co-cultura, mostramos que exosomes lançados de macrófagos interromper a expressão de proteínas de junção apertada nas células epiteliais brônquicas. Estes resultados sugerem que 1) conversas cruzadas entre inatas células imunes e estruturais através do vaivém exosomal contribuem para a resposta inflamatória e rompimento de barreira estrutural e 2) segmentação destes miRNAs pode fornecer uma nova plataforma para tratar ALI e SDRA.

Para induzir lesão pulmonar séptico, os ratos foram tratados com LPS intraperitoneal (15 mg/kg). Dentro de 24 horas da administração de LPS, neutrofílico influxo foi visto nos pulmões, conforme mostrado na figura 1A. Fluido de mouse BAL foi desenhado, seguido pelo isolamento e purificação de exosomes. Morfologia do LBA exosomes foi confirmada por microscopia eletrônica de transmissão (figura 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

Exosomes de lavagem broncoalveolar em lesão pulmonar séptico induzidas pelo lipopolissacarídeo

Os ratos expostos ao LPS intraperitoneal secretam exosomes no líquido de lavagem (BAL) bronco-alveolar que são embalados com miRNAs. Usando um sistema de co-cultura, mostramos que exosomes lançados no fluido BAL perturbará a expressão de proteínas de junção apertada nas células epiteliais brônquicas e aumentar a expressão de citocinas pró-inflamatórias que acentuam a lesão pulmonar.

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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