笔者要感谢莫里斯·史密斯和玛丽亚Talikka对稿件的审查。

在器官组织支气管文化模式1.文化注:再造组织模型购买的刀片是准备使用。备选地，培养物可以从原代细胞发展成为由卡普和同事16描述的例子。在收到在无菌包装组织，总是处理在无菌条件下的人体器官组织。 <ol><li>添加0.7预热（37℃）测定培养基到一个新的无菌的24孔板引擎盖下的每个孔中的溶液中。 </li><li>取出组织的包装罩下，并在组织培养插入物转移到新制备的24孔板（在步骤1.1中描述。上文）。 </li><li>维持和培养在培养箱中在组织中，在37℃（5％CO 2，90％湿度）。 </li><li>更换培养基，每2天。 </li><li>在一个介质改变，检查在光学显微镜下的组织，以确保它们它们contaminati在无和有没有泄漏介质中。 </li></ol> 2.香烟烟雾（CS）曝光用体外曝光系统<ol><li>在曝光之前实验三天中，组织培养的顶侧用200μl培养基洗涤。 </li><li>在暴露的一天，如果睫状跳动测量曝光之前计划，如在第3节中所述处理该组织为睫状打浆测量。 </li><li>预暖体外曝光系统的气候室和培养基本模块到37℃。与17.5毫升每行中的填充种植基地模块。 </li><li>从培养箱中取出的组织，并将其放置在通风橱下从培养板转移的组织培养插入到体外曝光系统的培养基的模块。此外，盖上玻璃盖的种植基地模块。 </li><li>转移组织在体外接触SYSTE的气候室米暴露于主流的CS。 </li><li>如果在体外暴露系统装有一个微量天平，设置了石英晶体微天平上运行的曝光来测量使用微量天平软件石英晶体烟雾的实时颗粒沉积之前。 <ol><li>更换晶体连接到稀释/分发系统中的每一行微量天平模块。 </li><li>微量天平模块连接到微量天平软件。在软件设置测量的刻度为零。 </li></ol></li><li>根据实验过程露出的组织（如在图1中如 ）。 注意:在此之前的曝光，参考香烟（3R4F）之间7至21天在受控条件下根据ISO标准3402 17调节至22±1℃和60±3％的相对湿度。 <ol><li>使用一个30端口转盘吸烟马赫生成香烟烟雾（ 例如 ，从3R4F香烟）国家统计局根据加拿大卫生部强烈的政权:抽香烟到一个标准的对接长度， 即约35毫米55毫升粉扑超过2秒，两次分8秒泵的排气时间。 注意:如果需要，稀释主流的CS，以使剂量没有毒性太大。在此处报道的结果，烟稀释至16％（体积/体积）。 </li><li>使用60％的加湿空气的对照样品（空气暴露）。 </li><li>暴露组织为6-7分钟（ 即 ，1香烟或空气暴露）在体外曝光系统</li><li>把组织放回培养箱中，在37℃（5％CO 2，90％湿度）1小时。 </li><li>重复步骤2.7.3和2.7.4三次。 </li></ol></li><li>在曝光实验结束时，停止微量天平软件。该软件将生成包含随着颗粒沉积的图形表示的所有测量值的文件。 </li><li>传回从种植B组织ASE模块到培养板中。将组织放回培养箱中，在37℃（5％CO 2，90％湿度），直到所述测量结果为在该特定曝光后的时间点的不同端点。 </li></ol> 3.测量睫状跳动注意:根据实验方案（图1），记录睫状打浆前曝光和后直接曝光。 <ol><li>从培养箱中取出的组织培养板中，并让它们在引擎盖下在室温30分钟以稳定该纤毛跳动。 </li><li>放置在组织中的光显微镜连接到CiliaMetrix软件下。 </li><li>记录睫状打浆的电影和频率（以赫兹为单位）。测量频率每3秒1分钟。 </li><li>返回组织回到培养箱中在37℃（5％CO 2，90％湿度）。 </li></ol> 4.跨上皮电阻（TEER）测量注意:TEER的测定进行3天前，用筷子电极（STX-2），连接到一个voltohmmeter无菌条件下罩下曝光后的48小时。 <ol><li>加入200μl培养基对人类支气管组织的顶端面。 </li><li>打开EVOMX机器上;用70％的乙醇溶液洗涤电极。 </li><li>用培养基清洗电极。 </li><li>通过不接触组织插入电极插入组织培养的根尖侧的介质测量的电阻（Ω）。 </li><li>重复步骤4.4 5倍，以获得一式五份的测量。 </li><li>测量后，轻轻地从使用抽吸的组织培养物的顶侧除去介质。 </li><li>返回的组织培养培养箱在37℃（5％CO 2，90％湿度）中培养。 </li></ol> 5.细胞色素P450（CYP）1A1 / 1B1 ActiviTY <ol><li>在研究开始前，被瓶的适当重建缓冲液中的整个内容转移到含有（按照制造商的说明）的荧光素检测试剂的琥珀色瓶中制备的荧光素检测试剂。 2毫升，在-20℃下等份商店最多1个月。 </li><li>为48小时后的曝光时间，孵育之后的CS暴露在37℃下将组织48小时。 </li><li>暖荧光素检测试剂至RT。 </li><li>组织孵育插入3小时或O / N与荧光素CEE基板（稀释至1:50）在基础培养基。 </li><li>转移50微升来自各组织培养基插入到一个96孔不透明白色光度计板在RT。 </li><li>添加50微升荧光素检测试剂至各孔以启动发光反应。 </li><li>孵育板在RT下20分钟。 </li><li>阅读使用发光的发光。 注:对于光度计，使用每孔0.25-1秒的积分时间为指针。 </li></ol> 6. RNA提取。 <ol><li>之前收集三维器官组织的插入，制备1）的盒用干冰，2）足够量的叶片（每3D刀片1），3）冷PBS无钙和氯化镁，4）1个25毫升玻璃移液管，和5 ）无菌玻璃针抽吸。 <ol><li>标签管充满了陶瓷珠均质固体细胞样本，并加入700μlQiazol的。 </li><li>打开吸机上。 </li><li>收集从培养箱三维器官组织插入物的板。 </li><li>用冷PBS在板洗每个插入的3倍。在洗涤之间，吸的PBS与玻璃针。在第三次洗涤后，吸用PBS和盖板，以防止插入件的干燥。 </li><li>用于从所述板的每个刀片，切出的插入物膜（3D组织刀片所在的），直到membrANE平躺下来的刀片。 </li><li>从叶片到均质管适当标记的转移组织（连同插入膜）并拧盖子上的管中，摇动和涡流它。 </li><li>冷冻在干冰和存储的样品在-80℃或继续萃取液中的RNA。 注意:在此之前的RNA的提取，根据随机设计（如果适用的话）标记所有管和列。准备根据制造商的建议的所有试剂，并采取了从-80℃冷冻样品并解冻它们在冰上直到它们被完全解冻。 </li></ol></li><li>同质化<ol><li>研磨样品均质仪对6000赫兹，持续45秒。如果样本不是正确匀浆并离开样品5分钟，在室温重复。 </li><li>加入140微升氯仿样品和旋涡10-15秒。 </li><li>从均质管转移上清到锁相管和动摇上thermoshaker为以1400rpm和RT 2分钟。 </li><li>在4℃下进一步温育在室温和样品2分钟离心样品在12000×g离心15分钟。 </li></ol></li><li> RNA沉淀，洗净，悬挂和净化QIAcube工作站。 <ol><li>传送在新的2ml管中的上层水相。 </li><li>加载所有必需的试剂提取机器人根据制造商的协议表，选择相应的协议，将洗脱体积（30微升），并运行提取的机器人。 </li><li>当运行完成后，取出转子适配器和立即保持洗脱管在4℃用于进一步的分析或在-80℃长期储存。 </li></ol></li><li>分离的RNA的质量控制<ol><li>确定根据制造商的说明，使用的是紫外线读取器的分离的RNA的量。 </li><li>检查RNA的质量和使用的RNA完整性微流体 - 凝胶为基础的芯片实验室和紫外线读取器，ACC奥尔丁制造商的说明。 </li></ol></li></ol> 7.芯片工作流程注意:下面描述的方法着重于方法Affymetrix基因芯片的高通量3&#39;IVT快车套件，其用于目标合成，用于对从所述制剂中的RNA，以使用一个复杂的起始的Affymetrix 3&#39;的基因表达盒的杂交液体处理系统（ 如 ，移液，稀释，分装，或整合）。 <ol><li>准备<ol><li>随机样本，以避免批量的影响。分批处理的样本的数目为从1到96，其中包括一个正（商业人类普遍参照RNA）和一个负（无RNA酶的水）每批次的控制。 </li><li>开始使用100ng的总RNA在3微升的无RNA酶的水的目标合成。对于100毫微克的总RNA，使用16小时的培养时间。 </li></ol></li><li> RNA扩增<ol><li>准备POLY-A RNA穗通过串行稀释聚-A RNA股票与聚-A控制稀释液控制解决方案: <ol><li>如需稀释1，准备2微升聚腺苷酸控制库存36微升稀释液。 </li><li>如需稀释2，准备2微升稀释1的98微升的稀释液。 </li><li>如需稀释3，准备4微升稀释2的196微升的稀释液。 </li><li>如需稀释4，在180微升的稀释液准备20微升稀释3。 </li></ol></li><li>淡化RNA酶自由水的RNA得到33.3纳克/微升的浓度。 </li><li>添加2微升的polyA对照溶液到3微升总RNA样品的直接在96孔板中。这个步骤是通过所述液体处理系统进行的。 </li><li>制备所述液体处理系统和目标合成。放置在机器人上的印版。将所有必需的试剂及实验室用品在甲板上。机器人将通过制备都是适宜启动过程Ëmastermixes和孵化，在自动遥控PCR块样品。 </li><li>一是质量控制检查:控制放大过程<ol><li>如果的aRNA的质量是可接受的，通过添加到每个样品8微升碎裂5X缓冲液（该步骤通过所述液体处理系统执行）执行的碎片的步骤。否则，执行前面的步骤再次从RNA开始。立即使用零散阿尔纳或储存未稀释的和零散的阿尔纳在-20°C（或-70℃下长期保存）。 </li><li>拿起随机12个样品的凝胶微流体系统的基础上盘和转让1微升检查碎片的效率进行第二次质量控制分析。 </li></ol></li></ol></li><li>杂交过程<ol><li>扫描芯片条形码，并根据指令制造商的软件注册每个样本。 </li><li>准备杂交组合为如下为一个单个反应并将其添加到33微升分散和标记的aRNA的:4.2微升对照寡核苷酸B2（为3nM），12.5微升20×真核生物杂交对照，加入25μl的100％DMSO中，125微升的2×杂交缓冲液和50微升的H 2 O代表的216.7微升的最终体积 注意:杂交，洗涤和处理样品之前扫描阳性和阴性对照。 </li><li>预湿用200μl的预杂交混合物中所含的杂交洗涤和染色试剂盒的阵列。 </li><li>放置在烘箱中于45℃杂交，芯片和60 RPM的10分钟。 </li><li>在此期间，变性在PCR块所述板（含有终鸡尾酒杂交混合物）在99℃下进行5分钟，45℃5分钟。 </li><li>从数组中取出的预杂交混合物，并将其用200μl的杂交混合液目标（每码片一个样本）取代。 </li><li>将数组复制到hybridization烘箱中在45℃下16小时（O / N）用60rpm的旋转速度。 </li></ol></li><li>洗涤和染色<ol><li>运行该软件菜单栏中的"射流"。在射流对话框中，选择感兴趣的（1 - 4）模块，然后选择Prime_450计划把所有模块。将管用于洗涤的缓冲液A中在瓶（200毫升）的瓶（400ml）中，并于洗涤缓冲液B，以及用于milliQ水中在瓶（500毫升）中。 </li><li>随后，按照液晶屏幕指示。抬起针和放置600微升染色鸡尾酒1（SAPE溶液混合）和含有微量离心管在位置＃1，＃2 600微升染色鸡尾酒2（抗体溶液混合），和900微升体列保持在位置＃3缓冲溶液。 </li><li>在O / N培养在烤箱后，从芯片吸出的鸡尾酒，并填写250微升洗涤液A.该芯片分配权芯片每个模块中，选择FS450-001协议和运行每个谟独乐寺，按照屏幕上的说明。这个过程持续90分钟。 </li><li>一旦液晶屏显示"弹出盒"的消息，取出芯片的，如果有任何气泡检查。如果存在气泡，完全填充阵列与阵列保持缓冲液中。申请贴到隔垫，以避免在扫描仪的任何泄漏和事先清洗微阵列窗口装载在扫描器。 </li></ol></li><li>扫描。 <ol><li>热身扫描仪。芯片加载到扫描仪的自动加载磁带机，并开始扫描。 注:在每个芯片〜12分钟，生成每个芯片.CEL文件。 </li><li>检查图像和对齐网格（如果需要的话），以将光点，以确定所述探针的细胞。 注:该数据集已提交Arrayexpress（登录代码= E-MTAB-1721）。 </li></ol></li></ol>对于影响评估8.基于网络的系统生物学分析<ol><li>微阵列数据的处理。 注:数据处理的ð评分方法采用R统计环境版本2.14落实。 <ol><li>打开一个R 18会话并加载AFFY 19 gcrma和affyPLM通过运行命令安装的软件包:库（AFFY）&gt;库（gcrma）&gt;库（affyPLM） </li><li>阅读运行命令的原始数据文件:data.affybatch &lt;-ReadAffy（"路径文件夹中存储的CEL文件"） </li><li>减去背景校正和位数师范使用gcrma包生成探针组的表达值，通过运行以下命令:eset.norm &lt;-gcrma（data.affybatch） </li></ol></li><li>质量控制。 <ol><li>产生RNA降解地块的命令:度&lt;-AffyRNAdeg（data.affybatch） </li><li>提取RNA降解斜率运行命令的系数:斜率= $度斜率</li><li>曲线的斜率系数，并确定可能的异常值。 </li><li>产生怒色和RLE地块运行以下命令:Pset的&lt; -fitPLM（data.affybatch）&gt; RLE值（Pset）&gt;怒色值（Pset）。 </li><li>如果确定某些生成的盒状图是异常值:一个数组被认为是离群如果至少有2定义见下文偏离其他阵列3 QC指标: - 度$斜率是由平均度坡度$不同 -怒色的情节:一个数组被视为异常，如果上四分位数低于0.95或高于1.05的下四分位数， 即 ，如果箱线图是完全高于1.05或低于全部0.95。 - RLE情节:一个阵列被认为是离群如果RLE分布的中值大于0.1或小于-0.1以下。 </li><li>如果阵列被确定为异常值，则丢弃，并且从步骤8.1.2重新开始。 </li><li>适合于数据的总的线性模型对所关注的具体对比（ 即 ，的"处理"和"控制"情况的比较）产生用于每个探针上的微阵列设置，它可以是furt原始P值她用LIMMA包的Benjamini-霍赫贝格程序进行调整。选择每个基因的探针在20叙述保持为代表的基因进行进一步的分析。 注:从实验设计中的阻塞因子（曝光板）占模型进行数据处理。 </li></ol></li><li>网络为基础的分析。 注意:如这里实施的方法中​​详细Martin等人所述。 （在修订BMC生物信息学）。 <ol><li>对于每个感兴趣的两两比较，从所计算的（log2-）启动网络（步骤8.2）下折的变化（处理与对照）对于每个基因。 </li><li>计算用L定义的网络中的有符号拉普拉斯矩阵L（I，J）= - 符号（I〜j）条瓦特（I，J），如果有重量的瓦特节点i和j之间的（I，J）的边缘， L（I，J）=度（i）是第i和L（I，J）= 0否则加权程度。权重w（I，J）等于1，如果i和j是在主链和瓦特（I，J）= 1 / N，如果i是一个骨干节点和j是其N neighb之一ORS的成绩单层。 </li><li>为f = L3-1L2Tx计算得分的骨干，其中L 3为L的子矩阵的骨干节点和L2为L的子矩阵的行对应于转录层节点和列到骨干节点</li><li>计算由其中所有边缘标志被反转骨干网定义的网络的拉普拉斯签署，Q。 </li><li>计算NPA得分的NPA = fTQf。 </li><li>计算的置信区间，骨干边缘置换p值和转录层置换p值。 注:（ 例如 ，样品之间的生物学变异在一个实验组）除了NPA分数的置信区间，其占了实验误差，同伴统计推导来描述NPA得分的特异性所描述的生物学在网络上。因为NPA是的倍数变化的二次形式，它的方差可以基于倍数变化估计V计算ariances。置信区间是用中心极限定理随后派生。两个置换试验实施21从而第一，评估，如果结果是特定于模型的底层证据（ 即 ，基因倍数变化），从而导致一个置换P值（表示为*○当P在图中 - 值&lt;0.05）。其次，评估网络"原因和结果"图层是否显著促进了网络扰动的幅度（图中记为K *当P值&lt;0.05）。 </li><li>考虑网络的影响通过处理，如果置信区间不包含零和两个排列的p值&lt;0.05。 </li><li>计算其被定义为在骨架贡献达80％的NPA得分的关键节点的主要节点。 </li></ol></li></ol>

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	<li>Pezzulo, A. 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=The+air-liquid+interface+and+use+of+primary+cell+cultures+are+important+to+recapitulate+the+transcriptional+profile+of+in+vivo+airway+epithelia.">The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia.</a> American Journal of Physiology - Lung Cellular and Molecular Physiolog. 300, L25-L31 (2011).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature reviews Molecular cell biolog. 8, 839-845 (2007).</li><li>Dvorak, A., Tilley, A. E., Shaykhiev, R., Wang, R., Crystal, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+airway+epithelium+air&#8211;liquid+cultures+represent+the+in+vivo+airway+epithelium+transcriptome.">Do airway epithelium air&#8211;liquid cultures represent the in vivo airway epithelium transcriptome.</a> American journal of respiratory cell and molecular biolog. 44, 465 (2011).</li><li>Wiszniewski, 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=Long-term+cultures+of+polarized+airway+epithelial+cells+from+patients+with+cystic+fibrosis.">Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis.</a> American Journal of Respiratory Cell and Molecular Biolog. 34, 39-48 (2006).</li><li>Balharry, D., Sexton, K., B&#233;ruB&#233;, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+approach+to+assess+the+toxicity+of+inhaled+tobacco+smoke+components:+nicotine,+cadmium,+formaldehyde+and+urethane.">An in vitro approach to assess the toxicity of inhaled tobacco smoke components: nicotine, cadmium, formaldehyde and urethane.</a> Toxicolog. 244, 66-76 (2008).</li><li>Mathis, C., 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=Human+bronchial+epithelial+cells+exposed+in+vitro+to+cigarette+smoke+at+the+air-liquid+interface+resemble+bronchial+epithelium+from+human+smokers.">Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers.</a> Am J Physiol Lung Cell Mol Physio. 304, L489-L503 (2013).</li><li>Cho, M. -. 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=A+bioluminescent+cytotoxicity+assay+for+assessment+of+membrane+integrity+using+a+proteolytic+biomarker.">A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker.</a> Toxicology in Vitr. 22, 1099-1106 (2008).</li><li>Stewart, C. E., Torr, E. E., Mohd Jamili, N. H., Bosquillon, C., Sayers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Differentiated+Human+Bronchial+Epithelial+Cell+Culture+Systems+for+Asthma+Research.">Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research.</a> Journal of Allerg. 2012, 11 (2012).</li><li>Blume, L. F., Denker, M., Gieseler, F., Kunze, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+corrected+transepithelial+electrical+resistance+(TEER)+measurement+to+quantify+rapid+changes+in+paracellular+permeability.">Temperature corrected transepithelial electrical resistance (TEER) measurement to quantify rapid changes in paracellular permeability.</a> Pharmazi. , 19-24 (2010).</li><li>Chhin, 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=Ciliary+beating+recovery+in+deficient+human+airway+epithelial+cells+after+lentivirus+ex+vivo+gene+therapy.">Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.</a> PLoS genetic. 5, e1000422 (2009).</li><li>Jiao, J., Meng, N., Wang, H., Zhang, L. <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+human+nasal+epithelial+cells+grown+as+explant+outgrowth+cultures+or+dissociated+tissue+cultures+in+vitro.">Comparison of human nasal epithelial cells grown as explant outgrowth cultures or dissociated tissue cultures in vitro.</a> Front Me. 7, 486-491 (2013).</li><li>Talbot, P. <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+assessment+of+reproductive+toxicity+of+tobacco+smoke+and+its+constituents.">In vitro assessment of reproductive toxicity of tobacco smoke and its constituents.</a> Birth defects research. Part C, Embryo today : review. 84, 61-72 (2008).</li><li>Port, J. 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=Tobacco+smoke+induces+CYP1B1+in+the+aerodigestive+tract.">Tobacco smoke induces CYP1B1 in the aerodigestive tract.</a> Carcinogenesi. 25, 2275-2281 (2004).</li><li>Hoeng, J., 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+network-based+approach+to+quantifying+the+impact+of+biologically+active+substances.">A network-based approach to quantifying the impact of biologically active substances.</a> Drug Discov Toda. 17, 413-418 (2012).</li><li>Iskandar, A. R., 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=Systems+approaches+evaluating+the+perturbation+of+xenobiotic+metabolism+in+response+to+cigarette+smoke+exposure+in+nasal+and+bronchial+tissues.">Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues.</a> Biomed Res In. 2013, 512086 (2013).</li><li>Karp, P. 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=An+in+vitro+model+of+differentiated+human+airway+epithelia.+Methods+for+establishing+primary+cultures.">An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures.</a> Methods in molecular biolog. 188, 115-137 (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ISO 3402: Tobacco and tobacco products -- Atmosphere for conditioning and testing. , (1999).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A Language and Environment for Statistical Computing. , (2011).</li><li>Gautier, L., Moller, M., Friis-Hansen, L., Knudsen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+mapping+of+probes+to+genes+for+Affymetrix+chips.">Alternative mapping of probes to genes for Affymetrix chips.</a> BMC Bioinformatic. 5, 111 (2004).</li><li>Hermida, 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=Confero:+an+integrated+contrast+data+and+gene+set+platform+for+computational+analysis+and+biological+interpretation+of+omics+data.">Confero: an integrated contrast data and gene set platform for computational analysis and biological interpretation of omics data.</a> BMC genomic. 14, 514 (2013).</li><li>Thomson, T. 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=Quantitative+assessment+of+biological+impact+using+transcriptomic+data+and+mechanistic+network+models.">Quantitative assessment of biological impact using transcriptomic data and mechanistic network models.</a> Toxicology and applied pharmacolog. 272, 863-878 (2013).</li><li>Teff, Z., Priel, Z., Gheber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forces+applied+by+cilia+depend+linearly+on+their+frequency+due+to+constant+geometry+of+the+effective+stroke.">The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke.</a> Biophysical journa. 94, 298-305 (2008).</li><li>Hilgendorf, C., 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=Caco-2+versus+Caco-2/HT29-MTX+co-cultured+cell+lines:+permeabilities+via+diffusion,+inside-+and+outside-directed+carrier-mediated+transport.">Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.</a> J Pharm Sc. 89, 63-75 (2000).</li><li>Sutto, Z., Conner, G. E., Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+airway+ciliary+beat+frequency+by+intracellular+pH.">Regulation of human airway ciliary beat frequency by intracellular pH.</a> The Journal of physiolog. 560, 519-532 (2004).</li><li>Zhang, X., 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=Similarities+and+differences+between+smoking-related+gene+expression+in+nasal+and+bronchial+epithelium.">Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium.</a> Physiological genomic. 41, 1-8 (2010).</li></ol>

这项研究是由美国菲利普莫里斯国际公司。

这里，我们已经证明人类器官支气管和鼻腔组织模型的适用性评估重复CS曝光的影响。作为替代动物试验，一些曝光系统的气溶胶暴露在体外毒理学评估被开发（ 例如 ，Vitrocell，Cultex，艾丽斯等）。这些曝光模块也可以用于空气中的污染物，空气中的颗粒，纳米颗粒等的毒理评估在这项研究中，我们使用了Vitrocell系统，可以容纳多达48个不同的样品同时，允许对较大规模的实验和下部之间的变异治疗。对于每一个气溶胶暴露在体外组织培养污染的风险仍然是一个重大的风险，其缓解需要一个小心处理的组织培养在整个实验。 电子期间微量天平测量颗粒沉积在实时xposure实验允许监视来自曝光系统中产生的CS剂量符合期望。以确保所测量的颗粒沉积的精度，设定安装在线测量正确的曝光之前是临界指数， 例如 ，规模设置为0。此外，由于微天平（厘米2）的区域之间的差的和组织培养插入物（0.33 平方厘米），我们调整最后计算在培养插入物的区域:只有33％在微量天平沉积的反映在培养插入物的实际沉积。 测量TEER的确定紧结屏障功能，并评估上皮层的破坏是一个相对容易的过程来实现，这是我们这里报告。然而，因为支气管和鼻腔组织模型包含黏液产生穿插杯状细胞，根尖洗涤需要TEER测定值之前必须执行urement。心尖​​洗涤是至关重要的，因为该粘液层的存在和厚度可偏压TEER的测量的可变性，在CS曝光的影响interferring。这个概念是在与什么是报告的Hilgendorf和同事，其中Caco-2细胞的渗透性受共培养与粘液产生杯状细胞系HT29 23的协议。粘液需要之前TEER测量被洗掉，因为在曝光前右心尖洗涤可以与组织反应的CS干扰。因此，进行测量曝光前三天，之前曝光不正确。 我们发现，CYP1A1 / CYP1B1活动可以从以下CS暴露的器官培养模型测量虽然活性仅略有增加CS。这个弱信号可由CYP1A1 / 1B1基板的较长的温育扩增（ 即 ，萤光素-CEE），例如用于24小时（数据未显示）。一个在目前的工作中的CYP活性测量的局限性是缺乏标准化到CYP蛋白质水平或向细胞计数，其可以被考虑为将来的研究，以确保对酶活性的改变不影响由任一蛋白水平或细胞计数的改变。 我们报道了CS暴露抑制纤毛击败两个鼻腔和支气管组织模型。类似的观察结果在不同的哺乳动物和非哺乳动物模型12完成。为睫状跳动测量，从而确保组织被处理，并且处理过的相似的方式是非常关键的，例如，如果介质变更实施，应适用于所有的样品。许特特和他的同事报道，pH值影响哺乳动物的纤毛跳动频率24。因此，比较以不同朱侃，陈万平/混合物，pH值处理的细胞之间跳动频率时调整，应考虑到最小化睫状打浆测量的可变性。此外，在该测量进行的温度也是关键的，因为睫状跳动的频率下降随着温度的降低。为了最大限度地减少由于这些变化，一个阶段顶培养箱，配备有温度，CO 2，湿度控制在这项研究中，使用的可变性。尽管这样，我们观察到，在睫状肌的CS曝光后的支气管组织跳动频率分别为高度可变（ 即 ，增加在一个插入件和减小另一个插入件），这表明睫状打浆是曝光后高度干扰的权利。与此相反，我们观察到了不存在可测量的睫状打浆频率中的CS曝光后的鼻组织，这表明鼻组织的反应更加敏感和一致。这是在协议与先前的出版物表明该鼻组织具有较低的容量来解毒作为与支气管25相比。 最后，我们发现，基因表达的器官支气管及鼻部组织模式受到CS分析可以证明CS对异生代谢产生影响。有趣的是，在异生素代谢所观察到的改变的器官支气管和鼻腔的体外模型暴露的CS表现得像是在体内的情况在吸烟者中更详细地在先前的出版物15中讨论。用于基因表达分析，使用机器人的仪器使高通量分析成为可能。此外，自动机器人装卸进一步增加的基因表达的结果的一致性和准确性。然而，快速采集的组织样品是至关重要的，以避免RNA提取过程中的RNA降解。的RNA加工和这里描述转录方法，也可以应用到体内的组织样本。

Lungs are directly and constantly exposed to inhaled air that may contain diverse toxicants such as pollutants and constituents of cigarette smoke (CS). Studying the impact of exposure to those toxicants on respiratory tissues is most informative when done in a manner that resembles in vivo exposure. Compared with the classical 2D immersed cell cultures (e.g., normal human bronchial epithelial cells (NHBE)), 3D organotypic tissue models better recapitulate the morphological, physiological, and molecular aspects of the human airway epithelium in vivo1,2: the 3D tissue models contain the diversity of the cell types observed in vivo, including differentiated epithelial cells, ciliated and non-ciliated cells, goblet cells, and basal cells. They have functional tight junctions and exhibit a mucociliary phenotype 1-3. Moreover, the cultures can be grown on a permeable porous membrane, in an air-liquid interface, allowing a direct exposure to aerosol at the apical side (whereas the basolateral side is immersed in culture medium) 3-5. Dvorak and colleagues reported that gene expression profiles of bronchial tissue models were similar to those obtained from human bronchial brushings 3. In addition, Mathis and colleagues showed that the responses of these tissue models to CS were similar to the differences observed between bronchial epithelial cells obtained from smokers and cells obtained from non-smokers 6. Finally, because the bronchial tissue models could be cultured for up to several months 4,5, they could potentially be used to examine the effects of long-term exposure of test items. 
Cytotoxicity assessments are common parameters measured following chemical insults or to assess the toxicity of specific compounds or mixtures. For instance, membrane integrity can be measured by a luminescent assay and allows the measurement of a dose-dependent cytotoxic effect on the cell culture 7. However, to assess pathophysiological effects of compounds at subtoxic concentrations, other parameters should be measured. For example, tissue integrity determined using the transepithelial electrical resistance (TEER) assay ensures the functionality of tight junctions and monitors the disruption of the epithelial layer 8,9. Ciliary beating frequency also allows the measurement of CS-related effects on respiratory tissues. A normal beating frequency for the cilia lining bordering the upper and lower respiratory tract is important to protect against airway infections 10. Each of the ciliated columnar epithelial cells of the respiratory epithelium has 200-300 cilia beating at a particular frequency to eliminate infectious agents or inhaled particulate matter trapped in the mucus released by interspersed goblet cells 11. CS contains chemicals that may inhibit ciliary beating 12, leading to a reduced protection of the respiratory tract. This work shows that ciliary beating can be measured in organotypic tissue models. This approach allows assessment of whether epithelial cells exhibit their normal function in the organotypic tissue culture. CS also activates xenobiotic metabolism responses in the respiratory tract to metabolize tobacco smoke constituents 13. The activity of the phase I xenobiotic metabolism enzymes, CYP1A1 and CYP1B1, of the tissue models can be measured. Additionally, as previously reported, global gene expression can be measured in the organotypic bronchial tissue models 6,14,15. A transcriptomic data and network-based systems biology approach is leveraged to assess the impact of CS on xenobiotic metabolism 15. 
The methodologies used to expose organotypic 3D bronchial and nasal tissue models to mainstream CS using an in vitro exposure system and to measure the tissue responses to this exposure compared to fresh air exposure (control) are detailed here.

<table><tbody><tr><td>MucilAir-human fibroblasts-bronchia</td><td>Epithelix S&amp;aacute;rl, Geneva, Switzerland</td><td>http://www.epithelix.com/content/view/122/19/lang,en/</td><td></td></tr><tr><td>MucilAir Culture Medium</td><td>Epithelix S&amp;aacute;rl, Geneva, Switzerland</td><td>http://www.epithelix.com/content/view/84/16/lang,en/</td><td></td></tr><tr><td>VITROCELL</td><td>VITROCELL systems GmbH, Waldkirch, Germany</td><td>http://www.vitrocell.com/index.php?Nav_Nummer=2&amp;amp;R=</td><td></td></tr><tr><td>3R4F reference cigarette</td><td>University of Kentucky</td><td>http://www2.ca.uky.edu/refcig/</td><td></td></tr><tr><td>30-port carousel smoking machine SM2000</td><td>Philip Morris, Int.</td><td></td><td></td></tr><tr><td>CiliaMetrix camera and software</td><td>La Haute &amp;Eacute;cole de Gestion (HESGE), Geneva, Switzlerland</td><td></td><td></td></tr><tr><td>Leica DMIL microscope&amp;nbsp;</td><td>Leica, Heerbrugg, Switzerland</td><td></td><td></td></tr><tr><td>LED light source</td><td>Titan Tool Supplies, Buffalo, NY</td><td></td><td></td></tr><tr><td>Chopstick Electrode STX-2</td><td>World Precision Instruments</td><td>http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/</td><td></td></tr><tr><td>EVOMXTM Epithelial Voltohmmeter&amp;nbsp;</td><td>World Precision Instruments</td><td>http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/</td><td></td></tr><tr><td>Luciferin Detection Reagent&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>MagNA Lyser Instrument&amp;nbsp;</td><td>Roche</td><td>http://www.roche.com/products/product-details.htm?region=us&amp;amp;type=product&amp;amp;id=66</td><td></td></tr><tr><td>chloroform&amp;nbsp;</td><td>Sigma-Aldrich</td><td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&amp;amp;region=</td><td></td></tr><tr><td>QIAcube&amp;nbsp;</td><td>Qiagen</td><td>9001882</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>http://www.nanodrop.com/</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer&amp;nbsp;</td><td>Agilent</td><td>http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106</td><td></td></tr><tr><td>Affymetrix GeneChip High throughput 3&amp;rsquo;IVT Express Kit</td><td>Affymetrix</td><td>http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit</td><td></td></tr><tr><td>Scanner 3000 7G&amp;nbsp;</td><td>Affymetrix</td><td>http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G</td><td></td></tr></tbody></table>

impact assessment of repeated exposure of organotypic 3d bronchial and nasal tissue culture models to whole cigarette smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

在下面所描述的效果，器官组织被初级人体上皮细胞的健康，不吸烟的隔离，高加索捐助者和成纤维细胞使用了15重组。 3D支气管和鼻腔组织模型 在体外的支气管和鼻腔器官模型在细胞水平上的人的呼吸道上皮（ 图2）类似。 CS曝光 器官支气管和鼻腔组织模型可反复暴露于主流的CS在空气 - 液体界面。用于这里示出的结果的烟雾暴露实验步骤示于图1。 纤毛跳动记录 纤毛打浆被记录在如图中视频空气暴露组织。 CS暴露与减少纤毛跳动频率有关:强峰周围观察在露出的假和前曝光4赫兹观察不到的CS曝光（ 图3）之后的任何时间。这降低频率会使纤毛跳动去除粘液和传染性病原体22效率较低。 TEER和CYP1A1 / 1B1活性测定 TEER测量48小时的最后曝光后，确保组织仍然正常工作。 CYP1A1 / 1B1活性也测定曝光的结束后48小时， 如图1中的CS-暴露组织中的TEER值没有显著不同，因为相比于空气暴露的组织，确认有烟雾没有重大的细胞毒作用在该浓度。在48小时后曝光，将组织的CYP1A1 / 1B1活性略有增加，这表明了组织防御机制以下的曝光（ 图4）的适度增加。 基因表达谱和基于网络的系统，双向易学的方法来学习CS暴露对异生代谢改变的影响 组织收集在48小时暴露后的时间点。接着，微阵列分析进行，以产生CS-基因表达谱和空气暴露组织。使用来自基因表达图谱杠杆基于网络的系统生物学方法对CS曝光上的生物外源性代谢的影响进行了研究，并如先前报道15。使用一个基于网络的系统生物学方法表明，在骨干节点的异型生物代谢网络模型的级别的CS曝光的影响（ 图5）之间， 在体外和体内数据集被很好地相关。与此相反，在每个单独的基因表达的改变程度，差的相关性， 在体外 （CS-暴露与空气接触），并在体内 （吸烟者与不吸烟者）之间观察到。 CS的重复暴露于器官支气管组织模型的图1示意图步骤缩写:CYP，细胞色素P450的; CS，香烟烟雾， TEER，跨上皮电阻。 <img alt="图2" src="/files/ftp_upload/52325/52325fig2.jpg" /> 图2.器官支气管和鼻腔上皮组织培养模型。卡通表示在空气-液体界面的支气管（A）和鼻（B）的组织培养插入物。 在体外模型中包含的苏木精-伊红染色的细胞的顶层所示纤毛细胞（ 下支气管，D为经鼻）如先前报道15。模型共培养与成纤维细胞是重要<html>用于生长和上皮细胞分化（用箭头表示）。染色气道系标记:P63（E支气管，女鼻）和MUC5AC（G支气管，H鼻）显示为此前15日报道， <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52325/52325fig3.jpg" /> 图3.纤毛跳动频率。纤毛跳动频率（CBF），测定在空气中暴露并在培养之前和曝光之后。下降的CBF的CS暴露后，看到（从两个刀片代表性结果显示为复制1（R1）和复制2（R 2））。 广告/ 52325 / 52325fig4.jpg"/&gt; 图4.测量CYP1A1 / CYP1B1活性和TEER的。左pannels表明在支气管（A）和鼻（B）的组织模型的CYP1A1 / CYP1B1活性。右侧面板指示在支气管（C）和鼻（D）的组织模型的TEER测定。装置±SD被示出。缩写:CS，香烟烟雾， CYP，细胞色素P450; TEER，跨上皮电阻; RLU，相对luminescense单位。 <img alt="图5" src="/files/ftp_upload/52325/52325fig5.jpg" /> 图5.基于网络的系统生物学方法对CS暴露在异生代谢的上下文中的影响的评估。的基因表达水平的变化被用来计算异型生物代谢网络模型的骨干节点的激活（A）的 </<html> STRONG&gt;报道在以前的出版物15。右边的图显示了骨干节点，也被称为量化的概念，"微分骨干网的价值，"采用NPA方法。蓝色椭圆表示的骨干节点的活性（ 即 ，功能层）和绿色球代表的基因的表达（ 即 ，转录层）。 CS暴露的器官组织支气管的影响（ 体外，CS-暴露与空气接触），在暴露后的48小时相比，这些吸烟对经支气管镜和鼻上皮收集burshing下鼻甲人支气管上皮（GSE16008 ）（ 在体内，吸烟者与非吸烟者）（B）中 。插图中，基因表达的相关性之间的体外和体内数据集变化。缩写:CS，香烟烟雾， NPA，网络扰动幅度。ove.com/files/ftp_upload/52325/52325fig5large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke at JoVE.com

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

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JoVE Journal

Bioengineering

11.1K 次观看.  Philip Morris Products S.A.. The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

视频: Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

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

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

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 ...

科研

医学

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

卷烟主流烟气中检测和清除气相自由基议定书

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

生物工程

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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

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 contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in 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 ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

免疫与感染

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

使用全身吸入系统在小鼠中暴露香烟烟雾

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

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

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 gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

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 ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

生物学

小鼠气道上皮细胞和慢性烟雾暴露的体外模型

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

作者聚焦:在 COPD 研究中研究慢性香烟烟雾暴露及其对气道上皮细胞影响的体外模型的开发和表征

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

香烟烟雾提取物刺激小鼠气道上皮细胞多组学分化和变化的体外模型

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

摘要

探索更多视频

此视频中的章节

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

卷烟主流烟气中检测和清除气相自由基议定书

电场控制的二维和三维环境中的神经祖细胞定向迁移

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

香烟烟雾对肺癫痫细胞伪多多多细胞的影响研究

使用全身吸入系统在小鼠中暴露香烟烟雾

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

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

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

香烟烟雾提取物刺激小鼠气道上皮细胞多组学分化和变化的体外模型

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

摘要

探索更多视频

此视频中的章节

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

卷烟主流烟气中检测和清除气相自由基议定书

电场控制的二维和三维环境中的神经祖细胞定向迁移

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

使用全身吸入系统在小鼠中暴露香烟烟雾

从切除的肺组织中分离支气管上皮细胞用于生物样本库并建立分化的气液界面培养物

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

用于真实、反复吸入空气颗粒以进行毒性测试的气液接口支气管上皮模型

香烟烟雾提取物刺激小鼠气道上皮细胞多组学分化和变化的体外模型

香烟烟雾对肺癫痫细胞伪多多多细胞的影响研究