Name: the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections
Uploaded: 2017-05-08T14:00:00
Description: Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

作者希望承认Vertex Pharmaceuticals和囊性纤维化研究创新奖，为R.Quinn和NIH / NIAID提供资金赠款1 U01 AI124316-01，一种用于治疗多重耐药性病原体的系统生物学方法。我们还要感谢UCSD本科机械工程高级设计课程的机械和航空航天工程系，以便与这项工作的工程方面进行协作。

1.股票的研制人工痰媒体<ol><li>创建一个5％粘蛋白的溶液。添加1.0克脱水猪胃粘蛋白的〜20毫升去离子水。高压釜中加入所得溶液。 注：粘蛋白消毒会破坏它的内在结构;其他方法来灭菌粘蛋白在其干燥形式包括紫外灭菌和辐射。这些方法还没有被广泛用于WinCF系统不过。 </li><li> 2.2克氯化钾添加到50毫升的去离子水，并允许溶解。 5.0克的NaCl添加至50mL的去离子水，并允许溶解。高压灭菌这两?...

<ol>
	<li>Quinn, R. 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=A+Winogradsky-based+culture+system+shows+an+association+between+microbial+fermentation+and+cystic+fibrosis+exacerbation.">A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation.</a> ISME J . 9, 1024-1038 (2015).</li><li>Quinton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis:+impaired+bicarbonate+secretion+and+mucoviscidosis.">Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis.</a> Lancet. 372 (9636), 415-417 (2008).</li><li>Harrison, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+ecology+of+the+cystic+fibrosis+lung.">Microbial ecology of the cystic fibrosis lung.</a> Microbiology. 153 (Pt 4), 917-923 (2007).</li><li>Caverly, L. J., Zhao, J., LiPuma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+lung+microbiome:+Opportunities+to+reconsider+management+of+airway+infection.">Cystic fibrosis lung microbiome: Opportunities to reconsider management of airway infection.</a> Pediatr pulmonol. 50, S31-S38 (2015).</li><li>Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+human+airway+microbial+ecology+reveals+a+pervasive+signature+for+cystic+fibrosis.">Quantitative analysis of the human airway microbial ecology reveals a pervasive signature for cystic fibrosis.</a> Sci Transl Med. 4 (153), 153ra130 (2012).</li><li>Willner, D., 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=Spatial+distribution+of+microbial+communities+in+the+cystic+fibrosis+lung.">Spatial distribution of microbial communities in the cystic fibrosis lung.</a> ISME J. 6 (2), 471-474 (2012).</li><li>Delhaes, 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=The+airway+microbiota+in+cystic+fibrosis:+a+complex+fungal+and+bacterial+community--implications+for+therapeutic+management.">The airway microbiota in cystic fibrosis: a complex fungal and bacterial community--implications for therapeutic management.</a> PloS one. 7 (4), e36313 (2012).</li><li>Rogers, G. B., 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=D.+Bacterial+diversity+in+cases+of+lung+infection+in+cystic+fibrosis+patients:+16S+ribosomal+DNA+(rDNA)+length+heterogeneity+PCR+and+16S+rDNA+terminal+restriction+fragment+length+polymorphism+profiling.">D. Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling.</a> J clin microbiol. 41 (8), 3548-3558 (2003).</li><li>Stenbit, A. E., Flume, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+exacerbations+in+cystic+fibrosis.">Pulmonary exacerbations in cystic fibrosis.</a> Curr Opin Pulm Med. 17 (6), 442-447 (2011).</li><li>Twomey, K. B., 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=Microbiota+and+metabolite+profiling+reveal+specific+alterations+in+bacterial+community+structure+and+environment+in+the+cystic+fibrosis+airway+during+exacerbation.">Microbiota and metabolite profiling reveal specific alterations in bacterial community structure and environment in the cystic fibrosis airway during exacerbation.</a> PloS one. 8 (12), e82432 (2013).</li><li>Carmody, L. 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=Changes+in+cystic+fibrosis+airway+microbiota+at+pulmonary+exacerbation.">Changes in cystic fibrosis airway microbiota at pulmonary exacerbation.</a> Ann. Am. Thorac. Soc. 10 (3), 179-187 (2013).</li><li>Worlitzsch, D., 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=Effects+of+reduced+mucus+oxygen+concentration+in+airway+Pseudomonas+infections+of+cystic+fibrosis+patients.">Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.</a> J. Clin. Invest. 109 (3), 317-325 (2002).</li><li>Cowley, E. S., Kopf, S. H., LaRiviere, A., Ziebis, W., Newman, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+Cystic+Fibrosis+Sputum+Can+Be+Chemically+Dynamic,+Anoxic,+and+Extremely+Reduced+Due+to+Hydrogen+Sulfide+Formation.">Pediatric Cystic Fibrosis Sputum Can Be Chemically Dynamic, Anoxic, and Extremely Reduced Due to Hydrogen Sulfide Formation.</a> mBio. 6 (4), e00767-e00715 (2015).</li><li>Sriramulu, D. D., L&#252;nsdorf, H., Lam, J. S., R&#246;mling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcolony+formation:+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung.">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54 (Pt 7), 667-676 (2005).</li><li>Quinn, R. 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=Biogeochemical+forces+shape+the+composition+and+physiology+of+polymicrobial+communities+in+the+cystic+fibrosis+lung.">Biogeochemical forces shape the composition and physiology of polymicrobial communities in the cystic fibrosis lung.</a> mBio. 5 (2), (2014).</li></ol>

与CF肺的微生物构成中含有种类繁多的生物，但在肺中的条件可能对什么样的微生物可以生存和发展13，15显著的影响。具体机制，通过这些条件的变化和它们对肺微生物的确切影响是目前通常不清楚。在该实验方法中，我们提出在模拟肺细支气管基于操纵化学条件变化微生物分析。 有协议的一些关键步骤。考虑到被分析的环境的微...

本手册中描述的方法称为WinCF系统1 。 WinCF的总体目标是提供一种能够模拟粘液填充肺支气管环境的实验装置。这将允许易感系统用粘液分泌过多的表型研究肺部疾病的微生物病原体，包括囊性纤维化（CF），慢性阻塞性肺病（COPD），哮喘等。该方法专门用于研究CF，其特征在于引起肺分泌物变稠且难以清除的突变，最终填充细支气管和其他具有粘液的小通道2 。肺中的这种阻塞抑制气体交换，因为吸入的空气不再能够到达许多肺泡，并且也为细菌定居3提供了栖息地。无法预防微生物生长肺过度粘液最终导致气道复杂的慢性感染的发展。这些社区含有多种生物，包括病毒，真菌，和类似的铜绿假单胞菌的细菌，都彼此5，6，7，8相互作用。在CF肺微生物的活性被认为是参与的症状称为肺部症状加重1，9，10，11耀斑。 WinCF能够围绕这些病情加重的微生物群落行为的研究，目前正扩大到作为一个基础的实验系统研究肺微生物生态学。传统上，加重已通过从肺部取出的样品的直接分析研究。许多混杂因素使微生物B的直接分析利用WinCF系统对肺部的行为进行挑战，除去许多这些因素，更直接地研究肺微生物组织的行为，从而更好地分析粘液塞细菌中的细菌活性。 WinCF系统提供了一种以有效模拟肺部环境的方式生长和分析细菌的方法。传统的肺细菌生长方法常常涉及在传统琼脂平板上培养样品。这些方法使样品对大气氧气开放，忽略了在粘液12,13接种的肺细支气管中发现的缺氧和经常缺氧的病症。在有氧条件下在琼脂上培养不像CF肺的环境，并且可能误导临床医生和研究人员关于他们试图治疗的病原体的行为。此外，琼脂平板上的细菌可用的营养物质与实际痰中可用的不相似，这通过使用人造痰介质（ASM）在WinCF中解决。如Sriramulu 等人的假单胞菌培养物所示。 14 ，ASM包括一组模拟可用于痰微生物的资源的组件，并且还可以复制痰的物理一致性。因为病肺具有特定的微生物组织，所以这种微生物的研究也应理想地发生在肺的特定条件下。 WinCF系统能够快速分析和轻松操作实验条件，以观察微生物的变化，类似于它们在实际肺细支气管中的发生。这种技术允许接种无数种相关样品，包括痰液，唾液，其他身体分泌物和纯的或混合的细菌培养物。实验设置的性质允许立即视觉解读微生物群落行为，旨在实现许多微生物和流行程序的下游应用。这些研究是重要的，因为细菌群落组成根据其环境的生理化学条件而变化。使用WinCF可以操纵介质的化学条件来分析对细菌活性的影响。例如，可以在接种样品之前改变培养基的酸度。孵育后，可以直接比较这些条件中的每一种的细菌活性，并且可以得出关于这些痰样品中的细菌如何响应不同pH值的行为的结论。在这里，我们概述了应用WinCF系统的程序和如何操作媒体化学来研究对肺微生物的影响的例子。

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Color-Coded Capillary Tubes</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">22-260943</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cha-seal Tube Sealing Compound</td>
			<td style="width:93px;">Kimble-Chase</td>
			<td align="right" style="width:119px;">43510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mucin from porcine stomach</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">M1778</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferritin, cationized from horse spleen</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;">F7879</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Salmon sperm DNA Sodium salt (sonified)</td>
			<td style="width:93px;">AppliChem Panreac</td>
			<td>A2159</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MEM Nonessential Amino Acids</td>
			<td style="width:93px;">Corning cellgro</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MEM Amino Acids</td>
			<td style="width:93px;">Cellgro</td>
			<td style="width:119px;">25-030-CI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Egg Yolk Emulsion, 50%</td>
			<td style="width:93px;">Dalynn Biologicals</td>
			<td style="width:119px;">VE30-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">P2157500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium Chloride</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">S271500</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">15mL centriguge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-666</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50mL centrifuge tubes with Printed Graduations and Flat Caps</td>
			<td style="width:93px;">VWR</td>
			<td style="width:119px;">89039-656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-150-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2.0mL microcentrifuge tubes</td>
			<td style="width:93px;">Corning</td>
			<td style="width:119px;">MCT-200-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

the wincf model - an inexpensive and tractable microcosm of a mucus plugged bronchiole to study the microbiology of lung infections

许多慢性气道疾病导致气道粘液堵塞。具有囊性纤维化的个体的肺是其粘液性细支气管产生微生物定植的有利栖息地的示例性情况。各种病原体在这种环境中相互茁壮成长，并驱使许多与CF疾病相关的症状。像任何微生物群落一样，其栖息地的化学条件对社区结构和动力学有重大影响。例如，不同的微生物在不同水平的氧气或其他溶质浓度下繁殖。在CF肺中也是如此，其中氧浓度被认为是驱动社区生理和结构。这里描述的方法被设计为模仿肺环境并以与它们引起疾病的方式更相似的方式生长病原体。然后使用这些微生物的化学环境的操作来研究化学物质肺部感染的疾病控制其微生物生态学。称为WinCF系统的方法是基于人造痰中和细毛细血管，旨在提供与存在于粘液细支气管中的氧梯度相似的氧梯度。操作化学条件，例如痰液的介质pH或抗生素压力，允许使用有色指示剂观察这些样品的微生物差异，观察气体或生物膜产生，或提取和测序每个样品的核酸含量。

在整个样本中引起的各种化学条件下微生物生长显着在某些情况下，更巧妙地在别人改变。在活动许多变化是视觉性的，只要潜伏期结束是显而易见的。在pH值操作的例子中，在整个pH值范围的试样，如图由温育后变得很明显多种因素变化很大。当无痰样品加入到培养基，温育48个小时后在整个pH值谱所表现出的唯一的变化是在培养基体积轻微蒸发（ 图9）产生?...

Watch this Scientific Journal Video about The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections at JoVE.com

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

粘液堵塞囊性纤维化的气道（CF）患者是微生物病原体茁壮成长的理想环境。手稿描述了用于研究CF肺微生物的环境中模仿其中它们引起的化学条件和疾病如何改变可驱动微生物动力学的新方法。

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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