Name: studying effects of cigarette smoke on pseudomonas infection in lung epithelial cells
Uploaded: 2020-05-11T14:30:00
Description: Watch this Scientific Journal Video about Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells at JoVE.com

这项工作部分得到了国家卫生研究院R01赠款HL125435和HL142997（对CZ）的支持。

1. 100% CSE 制备
<ol><li>将 10 mL 的无血清细胞培养基（BEAS-2B 细胞的 DMEM/F12;HSAEC 细胞的气道上皮细胞基质）绘制到 60 mL 注射器中。</li>
	<li>反向将适当修剪的 1 mL 移液器尖端连接到注射器的喷嘴中，作为用于容纳香烟 （3R4F） 的适配器。</li>
	<li>取出香烟的过滤器。将香烟连接到尖端适配器上，然后点燃香烟。</li>
	<li>将 40 mL 含烟空气吸入 10 mL 的无血清介质中。通过剧烈摇晃（每次绘制 30 s）将烟?...

<ol>
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细菌进入肺上皮细胞是细菌感染发病机制的关键一步。细菌进入细胞的过程可以分为以下三个步骤：第一，细菌接触并附着在上皮细胞表面使用其旗菌。其次，细菌要么进行内化，要么穿透细胞膜。最后，如果细菌成功逃脱细胞防御机制25，26，细菌复制和殖民细胞。观察肺微噬细胞细菌感染的方法早已发展起来，但对肺上皮细胞的了解有?...

吸烟影响全世界数百万人的公众健康。许多有害疾病，包括肺癌和慢性阻塞性肺病（COPD），据报与吸烟有关。,2吸烟会增加呼吸系统3、4、5中急性微生物,感染的易感性。此外，越来越多的证据表明，吸烟可以增强许多慢性疾病6、7、8,的,发病机制。例如，吸烟可能会增加病毒或细菌感染，导致慢性阻塞性肺病恶化9。在病因上导致慢性阻塞性肺病急性恶化的细菌病原体中，一种机会性克阴性杆菌病原体，伪多莫纳斯阿鲁吉诺萨，导致感染与不良预知和较高的死亡10，11。,11慢性阻塞性肺病的恶化通过加速病理进展使疾病恶化。除了抗症状管理12，没有有效的治疗COPD恶化。慢性阻塞性肺病的恶化会加剧病人的死亡率，降低生活质量，增加社会的经济负担。
呼吸道是一个开放的系统，不断受到外部存在的各种微生物病原体的影响。机会性细菌病原体通常在上气道中检测到，但有时在下气道14，15,中被发现。在动物模型中，在感染16后，只要1小时，就可以在阿尔维拉尔囊中检测到P.aeruginosa。作为一种主要的防御机制，免疫细胞，如巨噬细胞或嗜中性粒细胞，可消除气道中的细菌。肺上皮细胞作为第一个生理屏障，在宿主防御微生物感染方面具有独特的作用。肺上皮细胞可以调节微生物入侵、殖民化或复制，独立于免疫细胞17。上皮细胞中发现的一些分子，包括PPARg，发挥抗菌功能，从而调节细菌殖民化和复制在肺上皮细胞18。吸烟可能会改变分子，损害肺上皮细胞的正常防御功能19，20。19,20最近的研究表明，使用机器人吸烟装置21，22,将香烟烟雾直接暴露在肺上皮细胞中。然而，接触烟雾可以通过其他方式进行，包括应用 CSE。CSE 的制备是一种可重复的方法，具有其他细胞类型的潜在应用，包括间接暴露在香烟烟雾中的血管内皮细胞。
本报告描述了一种产生香烟烟雾提取物以改变肺上皮细胞中细菌负荷的协议。CSE增加 P.aeruginosa的细菌负荷，它可能有助于在COPD恶化中通常看到的细菌感染的复发。一种常规方法用于制备 CSE。肺上皮细胞，在他们的指数生长阶段，用4%的CSE治疗3小时。或者，单层培养的肺上皮细胞可以在空气-液体界面中直接暴露在香烟烟雾中。然后，CSE治疗的 细胞在 10的多重感染（MOI）上接受伪多多纳斯的挑战。细菌以特定的摇动速度传播，以确保其旗杆菌的形态保持不变，以保持其全部的侵入能力。根霉素用于杀死培养基中留下的细菌，从而减少细菌负荷的后续测定过程中的潜在污染。该协议还使用GP标签的伪多 monas，这已被作为研究不同模型中 伪多多纳斯 感染的有力工具。一个代表菌株 是P.氟森s 米古拉23。CSE治疗后感染程度或细菌负荷通过三种方式确定：带菌落计数的滴板方法，使用伪 多多 纳斯16S rRNA特异性底法的定量PCR，或感染荧光伪多多纳斯细胞的流细胞 仪。该协议是研究香烟烟雾对肺上皮细胞细菌感染的影响的简单且可重复的方法。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">50mL syringe</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">airway epithelial cell basal medium</td>
			<td style="width:148px;">ATCC</td>
			<td>PCS-300-030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Bacteria shaker</td>
			<td style="width:148px;">ThermoFisher Scientific</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">bronchial epithelial cell growth kit</td>
			<td style="width:148px;">ATCC</td>
			<td>PCS-300-040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Cell Counter</td>
			<td style="width:148px;">Bio-Rad</td>
			<td style="width:161px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:215px;">CFX96 Real-Time PCR System</td>
			<td style="width:148px;">Bio-Rad</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">High-Capacity RNA-to-DNA KIT</td>
			<td style="width:148px;">ThermoFisher Scientific</td>
			<td align="right" style="width:161px;">4387406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HITES medium</td>
			<td style="width:148px;">ATCC</td>
			<td style="width:161px;">ATCC 30-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">human BEAS-2B cells</td>
			<td style="width:148px;">ATCC</td>
			<td style="width:161px;">ATCC CRL-9609</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">human primary small airway epithelial cells</td>
			<td style="width:148px;">ATCC</td>
			<td style="width:161px;">ATCC PCS-300-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSRII flow cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Nikkon confocal microscope</td>
			<td style="width:148px;">Nikkon</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">OD reader</td>
			<td style="width:148px;">USA Scientific</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PCR primers</td>
			<td style="width:148px;">ITD</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Pseudomonas aeruginosa</td>
			<td style="width:148px;">ATCC</td>
			<td style="width:161px;">ATCC 47085</td>
			<td style="width:167px;">PAO1-LAC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Pseudomonas fluorescens Migula</td>
			<td style="width:148px;">ATCC</td>
			<td style="width:161px;">ATCC 27853</td>
			<td>P.aeruginosa GFP</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Research-grade cigarettes (3R4F)</td>
			<td style="width:148px;">University of Kentucky</td>
			<td style="width:161px;">TP-7-VA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy Mini Kit</td>
			<td style="width:148px;">Qiagen</td>
			<td align="right" style="width:161px;">74106</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Transprent PET Transwell Insert</td>
			<td style="width:148px;">Corning Costar</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Tryptic Soy Broth</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

studying effects of cigarette smoke on pseudomonas infection in lung epithelial cells

吸烟是导致肺气肿和慢性阻塞性肺病（COPD）的主要病因。吸烟也会促进呼吸系统细菌感染的易感性。然而，吸烟对人类肺上皮细胞细菌感染的影响尚未得到彻底研究。这里描述的是一个详细的协议，用于准备吸烟提取物（CSE），用CSE治疗人类肺上皮细胞，以及细菌感染和感染测定。CSE 是用传统方法准备的。肺上皮细胞在3小时用4%的CSE治疗，然后，在10的多重感染（MOI）感染伪多Pseudomonas多体。细胞的细菌负荷由三种不同的方法确定。结果表明，CSE增加肺上皮细胞的伪多多体负荷。因此，该协议提供了一种简单且可重复的方法，用于研究香烟烟雾对肺上皮细胞细菌感染的影响。

图用于说明图1 中的协议。肺上皮 BEAS-2B细胞用CSE治疗，并挑战伪多莫纳斯。培养基中的伪多多纳斯被添加的根霉素杀死，细胞接受滴板测定、伪多多体16SRNA的RT-qPCR检测和流动细胞切除术。与控制相比，CSE治疗在滴板方法中大大增加了细菌感染（图2）。相应地，CSE在HSAEC中受影响的细菌负荷（图3）。细胞生存?...

Watch this Scientific Journal Video about Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells at JoVE.com

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

这里描述的是一个协议，研究香烟烟雾提取物如何影响肺上皮细胞的细菌殖民化。

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 ...

该协议使用三种方法确定香烟烟雾是否影响肺上皮细胞中的伪多mons细菌负荷。此方法可以扩展到内皮细胞或其他细胞水龙头。本视频详细介绍了香烟烟雾提取、细菌感染和细菌负荷测定的准备情况。对于新用户来说，此过程很容易遵循。将10毫升无血清细胞培养剂放入60毫升注射器中。将修剪的窄端连接到注射器喷嘴上的一毫升移液器尖端，作为用于握住香烟的适配器。从香烟上取下过滤器，将香烟连接到适配器上。在进行检测前不超过30分钟，将香烟燃烧，并吸入40毫升含有空气的烟雾进入注射器。通过大力摇动注射器，将烟雾与介质混合。重复绘图过程，直到香烟完全烧坏，这需要大约 11 次绘制在大约 7 分钟内。要去除介质中的微生物和不溶性颗粒，请通过 0.22 微米过滤器进行过滤。然后，将介质转移到封闭的无菌管中。用选定的伪多多纳斯菌株接种一个试卵大豆汤或TSB螺旋钻板。在37摄氏度下孵育板。准备一个含有20毫升TSB的管子，以5%甘油作为碳源。从螺旋板中收集污迹，然后接种TSB。在 200 RPM 的 37 摄氏度摇床中孵育伪单子悬浮液约 1 小时，直到 600 纳米的光学密度为 0.6。培养手稿中描述的肺上皮细胞后，向细胞中加入一毫升0.25%的三辛。大约五分钟后，当细胞完全脱离板底时，加入10毫升的完整高度介质，以中和三辛。将细胞转移到15毫升管。然后，在摄氏四度和300倍G.下离心5分钟，取出并丢弃上一代。然后，用10%FBS将细胞重新悬浮成高地介质的毫升。移液10微升的上皮细胞悬浮到一个新的板。使用自动细胞计数器获取细胞浓度。每毫升以每毫升两毫升的中等浓度将肺上皮细胞以三倍十倍至第五细胞的浓度镀块。一夜之间孵育盘子。当细胞达到约80%汇合时，用"高度"介质替换介质，外加1%FBS。然后，在井中加入4%CSE，并孵育板3小时。在经过 CSE 治疗的肺上皮细胞的每个井中添加一个伪多体。然后，用5%的二氧化碳在37摄氏度下孵育板一小时。接下来，用两毫升新鲜高地介质代替六井板中的介质，每毫升根霉素含有 4%CSE 和 100 微克。在37摄氏度下用5%的二氧化碳孵育板一小时后，从井中吸出介质。用两毫升的冷PBS清洗两次经过基因霉素处理的细胞。然后，可以通过滴板方法、qRT-PCR 或流式细胞仪检测肺上皮细胞中的细菌负荷。在肺上皮细胞的每个井中加入一毫升细胞解液缓冲液。修复细胞的连续稀释，范围从1到10到1到10，000。然后接种TSB螺旋板。经过16个小时的孵育，通过计算细菌菌落的数量，确定肺上皮细胞中存在多少菌落形成单位。在肺上皮细胞的每个井中加入0.35毫升的硫化盐解液缓冲液。使用单元格刮刀收集单元格。将落酸移入微离心管中，与移液器轻轻混合。将 0.35 毫升新鲜准备的 70% 乙醇加入到 lysate 中，并混合好。将所有样品转移到放置在两毫升收集管中的旋转柱上。在10，000倍的G和20至25摄氏度的收集管中离心30秒。丢弃收集管中的缓冲液，用 0.7 毫升洗涤缓冲液清洗柱。将柱子在10，000倍G下离心30秒。用 0.5 毫升缓冲液清洗柱两次。在 10，000 次 G 下重复离心两分钟。将柱放入新的 1.5 毫升收集管中，并加入 30 至 50 微升无 RNase 水。在10，000次G下离心管一分钟后，收集流经并测量RNA浓度。为了准备20微升逆转录反应，使1微克的总RNA与10微升的反应缓冲液，一微升的逆转录酶和RNase无水。根据制造商的协议，在37摄氏度下进行逆转录反应1小时，然后在95摄氏度下进行5分钟。然后将每个 cDNA 样品的一个微升与包含网络染料的主混合物的五微升混合，以及每个 200 纳米摩尔特异性底法的一个微升。加入水，总体积为20微升。根据制造商的建议执行 PCR 分析。然后使用比较 CT 方法来确定表达式。在 BEAS-2B 细胞中，当细胞用 4%CSE 治疗 3 小时时，生存能力没有显著变化。一个小时的假多单体感染也没有实质性影响生存能力。最后，他们利用基因霉素在培养基中杀死伪多莫纳斯，对细胞生存能力没有统计学上显著的影响。根据滴板法测量，BEAS-2B细胞的细菌感染通过CSE治疗显著增加。HSAEC的CSE治疗也是如此。使用RT-PCR对经过采为治疗的 BEAS-2B 细胞的细菌感染进行评估，以量化存在 16S rRNA 的量，这表明伪多多纳斯 aeruginosa 的增加。使用 CSE 治疗并感染伪多荧光米古拉的 BEAS-2B 细胞在 509 纳米流式细胞学分析。结果表明，CSE治疗会增加细菌感染。荧光显微镜的结果表明，在伪多多纳斯荧光米古拉感染实验中，GFP标记细菌与 BEAS-2B 细胞共同定位于。细胞可能暴露在香烟烟雾与机器人吸烟，这可能模仿人类的行为，并使得可以直接研究香烟烟雾对肺上皮细胞的影响。在肺感染的背景下，这种技术可以用来探索其他细胞水龙头上的香烟烟雾。所述方法是开发有效疗法的基础，以对抗吸烟后肺损伤和C0PD额外

Research

JoVE Journal

Immunology and Infection

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

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Application of a Mouse Ligated PeyerÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Pseudomonas aeruginosa Induced Lung Injury Model

Pseudomonas aeruginosa Induced Lung Injury Model

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

对白细胞从流程的分析招聘基质细胞的影响

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

香烟烟雾对肺 癫痫细胞 伪多多多细胞的影响研究

香烟烟雾对肺癫痫细胞伪多多多细胞的影响研究