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

作者宣布没有利益冲突。

总的协议需要2天, 包括第一天的败血症诱导和分离从动物的球液, 第二天的外来体隔离从老鼠 BALF。所有程序都已由亚特兰大 VA 医疗中心的机构动物护理和使用委员会审查和批准。
1. 小鼠急性脓毒性肺损伤模型
<ol><li>使用 6-8 周-老雄性野生型 C57BL/6J 小鼠 (重量 20-22 克) 的动物模型, 并执行所有程序使用无菌技术和仪器。高压釜所有手术器械, 包括镊子和剪刀, 20 分钟121摄氏度?...

<ol>
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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. 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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. 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小鼠疾病模型通常用于评估特定基因的生理功能, 并降低实验费用2。这里描述的急性脓毒性肺损伤模仿人与 ARDS 的炎症反应。该模型与研究分子发病机制、生物标志物的发展以及测试潜在的新疗法5有关。
共培养系统与体内研究有关, 它是在对体外系统23、30中的生物分子 (如 RNA、DNA、脂质?...

阿里和 ARDS 是一种危及生命的呼吸衰竭, 其严重的低氧血症由非心源性肺水肿引起, 每年全球约有100万人感染1。急性呼吸窘迫综合征的病因包括感染或吸入对肺部的直接损伤和各种间接侮辱。在过去的十年中, 人们对 ards 的分子发病机制有了越来越多的了解, 但是, 针对 ards 的特定靶向治疗还有待开发2,3。
建立了几种急性肺损伤动物模型, 为人类研究的实验治疗提供了一座桥梁4,5。通常使用的模型包括油酸、细菌、LPS 和博莱霉素的本地安装。其他方法包括缺血再灌注、盲肠结扎穿刺、机械通气引起的伸展损伤、高氧或系统性细菌的管理和 LPS5。这些模型提供了一个有用的生物学系统来测试临床假说和发展潜在的治疗方法。为了模拟人 ARDS, 动物模型应重现炎症和急性损伤的上皮和内皮细胞的缺陷, 在屏障功能的肺部。
外来体是 20-200 nm 直径的膜泡, 其分子含量包含蛋白质、DNA、RNA 和脂质, 并通过分子组成转移促进组织微环境中的细胞间通信。外来体由多种类型的细胞分泌, 如内皮细胞、上皮细胞、平滑肌细胞和肿瘤细胞, 并存在于人体体液中。研究表明, 外来体调节免疫细胞和基质细胞在传染性和无菌炎症性疾病中的交叉交谈, 其异常释放似乎受到各种自然和实验性刺激的调节, 在生理和病理过程6。这种通信网络可能在肺部疾病的发病机制中发挥重要作用, 并可能影响病理生理学进展7,8。作为 18-22 核苷酸非编码 rna, miRNAs 存在于组织和体液, 血浆, 血清和调制 mRNA 表达在后平移水平9,10。
包装 miRNAs 在外来体影响分化和多种类型细胞的功能, 过多的水平是与各种疾病, 包括癌症, 肺部疾病, 肥胖, 糖尿病和心血管疾病, 11, 12,13,14,15,16。进入接受细胞和穿梭 exosomal miRNAs 促进细胞间通讯, 改变微环境的止血17,18。急性肺损伤是一个复杂的过程, 涉及多细胞类型与广泛的细胞间通讯通过外来体 8.miR-155 和 miR-146a 共享共同的转录调节机制, 并有助于炎症反应和免疫耐受性19,20。最近的研究表明, 两种调节炎症反应通过 exosomal miRNAs 穿梭在免疫细胞之间的21。然而, exosomal miRNAs 对内毒素的肺泡反应的调节作用的分子机制尚不清楚, 毫无疑问, 潜在的临床相关性和转化暗示值得进一步研究。
共用培养模型用于定义复杂环境中特定细胞类型的相互作用, 如炎症和癌症22,23。这些平台提供了一个替代的策略, 以询问细胞类型之间的交叉谈话, 特别是免疫和结构细胞。
内毒素的气管内、雾化、腹腔或全身管理被广泛用于诱发实验性肺损伤24,25,26, 并显示诱导上皮和内皮渗透性缺陷。在这里我们用腹腔内毒素诱导小鼠急性肺损伤的脓毒性模型。在内毒素腹腔内24小时内, 肺部出现通透性缺损, 并招募炎症细胞。此外, 我们还表明, 外来体从 miRNA-155 和 miR-146a, 和外来体从球液诱导促炎细胞因子表达的受体上皮细胞, 包括 IL-6 和 TNF α。这些数据是首次表明, exosomal miRNAs 是分泌的急性脓毒症肺损伤模型。

<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

急性肺损伤 (ALI) 和急性呼吸窘迫综合征 (ARDS) 是一种不同种类的肺部疾病, 持续高发病率和死亡率。阿里的分子发病机制得到了更好的界定;然而, 由于疾病的复杂性质, 分子疗法还有待开发。在这里, 我们使用脂多糖 (LPS) 诱导小鼠急性脓毒性肺损伤模型, 描绘了外来体在炎症反应中的作用。使用这个模型, 我们可以表明, 暴露于腹腔内 LPS 分泌外来体的小鼠从肺部的支气管肺泡灌洗 (球) 液中, 包裹着 miRNA 和细胞因子, 调节炎症反应。进一步使用共培养模型系统, 我们表明, 外来体释放的巨噬细胞扰乱紧张连接蛋白的表达在支气管上皮干细胞。这些结果表明, 1) 通过 exosomal 穿梭的先天免疫和结构细胞之间的交叉交谈, 有助于炎症反应和破坏的结构屏障和 2) 针对这些 miRNAs 可能提供一个新的平台, 以治疗阿里和 ARDS。

为诱发脓毒性肺损伤, 小鼠采用腹腔内 LPS (15 毫克/千克) 治疗。在 LPS 管理的24小时内, 中性的涌入出现在肺部, 如图 1A所示。绘制了小鼠球液, 然后分离纯化了外来体。用电子透射显微镜 (图 1B) 证实了 BALF 外来体的形态学。
从纯化外来体中检测 miRNA-155 和 miR-146a 的选择性促炎 miRNAs ?...

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

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

暴露于腹腔内的 LPS 分泌外来体的小鼠支气管肺泡灌洗 (球) 液, 包装与 miRNAs。使用共培养系统, 我们表明, 外来体释放的球液扰乱支气管上皮细胞紧密连接蛋白的表达, 并增加支持炎症细胞因子的表达, 加重肺损伤。

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...