Name: isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface
Uploaded: 2011-02-21T17:00:00
Description: Watch this Scientific Journal Video about Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface at JoVE.com

我们感谢技术援助和博士Shivraj Tyagi埃梅卡Ifedigbo宝贵的专业知识。我们也感谢哈佛NeuroDiscovery中心与显微镜的帮助。这项工作是支持部分由美国心脏协会授予09PRE2250120 Predoctoral林Hilaire指出，与美国国立卫生研究院拨款，以R01 - HL60234，R01 - HL55330，R01 - HL079904，授予AMK的财。

整个协议需要2天从动物组织中的细胞，细胞增殖的5-10天，并在气 - 液界面的细胞分化的一个额外的10-14天的隔离。一个额外的一天所需的细胞暴露和样品的收获。 1。鼠标气管，支气管上皮细胞（MTEC）的分离。 注意：下面描述的所有程序进行了审查和批准机构的动物护理和使用布里格姆与妇女医院/哈佛医学院区委员。 在开始之前： </p...

<ol>
	<li>You, Y., Richer, E. J., Huang, T., Brody, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+mouse+tracheal+epithelial+cells:+selection+of+a+proliferative+population.">Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 283, L1315-L1320 (2002).</li><li>Davidson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+epithelial+cells:+isolation+and+culture.">Murine epithelial cells: isolation and culture.</a> J. Cyst. Fibros. 2, 59-62 (2004).</li><li>Davidson, D. J., Kilanowski, F. M., Randell, S. H., Sheppard, D. N., Dorin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primary+culture+model+of+differentiated+murine+tracheal+epithelium.">A primary culture model of differentiated murine tracheal epithelium.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 279, L766-L778 (2000).</li><li>Rabe, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al.;+Global+Initiative+for+Chronic+Obstructive+Lung+Disease.+Global+strategy+for+the+diagnosis,+management,+and+prevention+of+chronic+obstructive+pulmonary+disease:+GOLD+executive+summary.">et al.; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary.</a> Am. J Respir. Crit. Care Med. 176, 532-555 (2007).</li><li>Macnee, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+chronic+obstructive+pulmonary+disease.">Pathogenesis of chronic obstructive pulmonary disease.</a> Clin. Chest Med. 28, 479-513 (2007).</li><li>Tuder, R. M., Yoshida, T., Arap, W., Pasqualini, R., Petrache, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art.+Cellular+and+molecular+mechanisms+of+alveolar+destruction+in+emphysema:+an+evolutionary+perspective.">State of the art. Cellular and molecular mechanisms of alveolar destruction in emphysema: an evolutionary perspective.</a> Proc. Am. Thorac. Soc. 3, 503-510 (2006).</li><li>Yao, H., Rahman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+on+the+role+of+inflammation+in+COPD+and+lung+cancer.">Current concepts on the role of inflammation in COPD and lung cancer.</a> Curr. Opin. Pharmacol. 9, 375-383 (2009).</li><li>van der Toorn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+irreversibly+modifies+glutathione+in+airway+epithelial+cells.">Cigarette smoke irreversibly modifies glutathione in airway epithelial cells.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 293, L1156-L1162 (2007).</li><li>Slebos, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+localization+and+function+of+heme+oxygenase-1+in+cigarette+smoke-induced+cell+death.">Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death.</a> Am. J. Respir. Cell Mol. Biol. 36, 409-417 (2007).</li><li>Kim, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+proteins+regulate+cigarette+smoke+induced+apoptosis:+protective+role+of+heme+oxygenase-1.">Autophagic proteins regulate cigarette smoke induced apoptosis: protective role of heme oxygenase-1.</a> Autophagy. 4, 887-895 (2008).</li><li>Chen, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egr-1+regulates+autophagy+in+cigarette+smoke-induced+chronic+obstructive+pulmonary+disease.">Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.</a> PLoS ONE. 3, e3316-e3316 (2008).</li><li>Okuwa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+micronucleus+assay+for+cigarette+smoke+using+a+whole+smoke+exposure+system:+A+comparison+of+smoking+regimens.">In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens.</a> Exp Toxicol Pathol. , (2009).</li><li>St-Laurent, J., Proulx, L. I., Boulet, L. P., Bissonnette, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+in+vitro+models+of+cigarette+smoke+exposure.">Comparison of two in vitro models of cigarette smoke exposure.</a> Inhal. Toxicol. 21, 1148-1153 (2009).</li><li>Watson, A. M., Benton, A. S., Rose, M. C., Freishtat, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+alters+tissue+inhibitor+of+metalloproteinase+1+and+matrix+metalloproteinase+9+levels+in+the+basolateral+secretions+of+human+asthmatic+bronchial+epithelium+in+vitro.">Cigarette smoke alters tissue inhibitor of metalloproteinase 1 and matrix metalloproteinase 9 levels in the basolateral secretions of human asthmatic bronchial epithelium in vitro.</a> J Investig. Med. 58, 725-729 (2010).</li><li>Rennard, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cigarette+smoke+in+research.">Cigarette smoke in research.</a> Am. J. Respir. Cell Mol. Biol. 31, 479-480 (2004).</li><li>Shapiro, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smoke+gets+in+your+cells.">Smoke gets in your cells.</a> Am. J. Respir. Cell Mol. Biol. 31, 481-482 (2004).</li></ol>

协议描述小鼠气管上皮细胞的分离，是改编自你等的协议 ，1，和其他修改2-3。至于任何协议，描述细胞的隔离，最关键的环节，以避免细菌或真菌病原体的污染，采用严格的无菌操作技术。第二个关键步骤，以避免成纤维细胞污染的文化，可避免的气管仔细解剖，和阴性选择步骤1.19。文化监控，洗净，和文化传媒补充隔日最初的72小时孵化后，应充分区分文化在大约两个?...

*吸烟机是一个定制的设计和制造14“X14”X20“双墓室和水套光色调清楚地证明1 / 2”厚的聚碳酸酯的Lexan室温度控制，水位传感器香烟烟雾暴露控制关闭系统。一个吸烟/膨化机是安装一个变量的膨化率的香烟。当单位在使用时，它模仿孵化器中的温度，湿度和二氧化碳控制系统中的意义。该系统包括：（一）一个定制的双室/水套单元，保持一个控制的环境中进行组织培养实验。 （II）数字重型，高精度双泵水温水位传感器和温度控制（三​​）香烟与吸烟单位膨化泵循环体系。 （四）泵循环传感器控制率循环仪（IV）不锈钢高精密线滤波器持有。 （五）安装一个可拆卸的盖子“大小的轴向均匀香烟烟雾混合风扇（六）一个中等大小的水的循环与安装支架水浴（七）11 / 2 1 / 2”厚的有机玻璃托盘的括号粉扑泵和烟灰收藏家持有。 这描述的机器可以取代类似市售一种从谢系统（如吸烟<a href="http://www.tse-systems.com">机www.tse -</a> systems.com） 。

isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface

肺上皮细胞可以分离出小鼠的呼吸道及气 - 液界面（ALI）的培养作为一个差异化的呼吸道上皮模型。协议是描述卷烟主流烟雾（CS）隔离和揭露这些细胞，上皮细胞的反应，以研究为CS暴露。该协议由三部分组成：隔离的呼吸道上皮细胞从小鼠气管，气 - 液界面（ALI）作为完全分化的上皮细胞，这些细胞的培养，这些细胞在文化和校准主流CS交付。 ALI的文化系统允许的条件下，更接近于他们比普通的液体培养系统的生理设置的呼吸道上皮细胞的文化。接触到CS分子和肺癌细胞反应的研究是了解环境空气污染对人体健康的影响的关键组成部分。最终，在这方面的研究结果可能有助于了解慢性阻塞性肺疾病（COPD），及其他与烟草有关的疾病，这​​代表了重大的全球性健康问题的病因。

Watch this Scientific Journal Video about Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface at JoVE.com

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

从小鼠的呼吸道和肺上皮细胞可以分离，气液界面的差异化的呼吸道上皮模式培养。协议是分离，培养，这些细胞暴露卷烟主流烟雾，为了研究这种环境毒素的分子反应。

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Research

JoVE Journal

Medicine

Experimental Human Pneumococcal Carriage

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

科研

医学

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

比较电子烟和烟雾在小说中的影响<em&gt;体内</em&gt;曝光系统

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

转录组水平上人类原发性肠系膜动脉内皮细胞的分离和分析

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the ...