这项研究得到了中国国家自然科学基金 （82090013） 的支持。

整个方案需要 44 天，包括从小鼠气管中分离的气道上皮细胞的制备 1 天、细胞增殖的 15 天和气液界面 CSE 刺激的 28 天。所有实验动物均饲养在首都医科大学动物实验中心的 SPF 屏障动物室，并经首都医科大学动物实验与实验动物伦理委员会 （AEEI-2020-100） 审查批准，符合 ARRIVE 指南7 的要求。
1. 从小鼠气管中分离原代小鼠气道上皮细胞
<ol><li>制备<ol><li>通过将 1 mL 50x 添加剂和 0.05 mL 氢化可的松原液与 48.95 mL 扩增基础培养基混合来制备完全扩增培养基。储存在 4 °C 并在 4 周内使用。</li>
		<li>将 7.5 mg 蛋白酶和 5 mg 脱氧核糖核酸酶 I 溶解在 5 mL Ham's F-12 中，制备蛋白酶溶液。过滤消毒并在 4 °C 下储存，并在 4 周内使用。</li>
		<li>通过将 0.05 mL 100x 青霉素/链霉素和 0.01 mL 500x 庆大霉素/两性霉素添加到 5 mL 完全扩增培养基、PBS 和蛋白酶溶液（100 U/mL 青霉素、100 U/mL 链霉素、10 μg/mL 庆大霉素和 0.25 μg/mL 两性霉素 B）中来制备抗生素溶液。储存在 4 °C 并在 4 周内使用。</li>
		<li>对手术器械进行消毒，并准备用于细胞分离的生物安全柜。同时，确保标准细胞培养设备已准备好使用。</li>
		<li>通过将大鼠尾胶原蛋白（100 μg/mL 在 0.02 N 乙酸中）添加到 100 mm 培养皿和 24 mm transwell （0.05 mL/cm2） 中来制备大鼠尾部胶原蛋白涂层的培养皿。在紫外线下打开过夜进行消毒。</li>
	</ol></li>
	<li>原代小鼠气道上皮细胞的分离<ol><li>使用 C57BL/6 小鼠 （6-8 周龄，雄性，SPF 升高） 进行气管上皮细胞分离。用 1% 戊巴比妥钠溶液过量（约 200 mg/kg）安乐死。使用五只小鼠以获得足够的细胞产量。</li>
		<li>将小鼠浸入 75% 乙醇溶液中对小鼠进行绝育，不要将鼻子和嘴巴浸入酒精中，以防止酒精流入气管。</li>
		<li>解剖鼠标。<ol><li>使用手术刀片和镊子，这是一套用于切割和打开从小鼠下颌到腹腔的表皮的装置。</li>
			<li>使用另一套手术器械撕开两侧的甲状腺，去除气管、周围肌肉组织和食道之间的连接。</li>
			<li>之后，小心切开胸部，用镊子深入胸腔，取出整个肺，找到气管末端。</li>
		</ol></li>
		<li>处理气管。<ol><li>从甲状软骨到气管支气管支气管分支切出气管，并将其放入含有四种抗生素的预冷膨胀培养基中。</li>
			<li>摇动管子，尽可能多地冲洗掉气管表面的血液，将其保持在冰上，然后开始为下一只小鼠进行手术。</li>
		</ol></li>
		<li>将所有收集的气管转移到含有四种抗生素（200 U/mL 青霉素、200 U/mL 链霉素、20 μg/mL 庆大霉素和 0.5 μg/mL 两性霉素 B）的预冷 PBS 缓冲液中，以便在消化前进行预处理。</li>
		<li>使用一套手术器械去除气管表面的凝块和其他组织，然后将它们切成 1 cm2 的大小。</li>
		<li>将气管组织在蛋白酶溶液中于 37 °C 孵育 40 分钟。</li>
		<li>40 分钟后，摇动试管数次，并使用 40 μm 细胞过滤器去除剩余的组织。在装有 5 mL 扩增培养基的过滤器上冲洗切碎的气管，在 4 °C 下以 400 x g 离心细胞悬液 5 分钟，然后弃去上清液。</li>
		<li>将获得的细胞重悬于 8 mL 扩增培养基中。</li>
		<li>使用台盼蓝和血细胞计数器计数活细胞。</li>
		<li>将 P0 细胞悬液接种在预涂有大鼠尾胶原的 100 mm 培养皿（1 x 104 个活细胞/cm2）上，并在 37 °C、5% CO2 的培养箱中培养细胞。</li>
	</ol></li>
</ol>2. 原代小鼠气道上皮细胞的扩增培养和传代
<ol><li>每 3 天更换一次 P0 细胞培养物的培养基，直到细胞达到 70%-80% 汇合度，通常在 6 天内。此时，小鼠气管上皮细胞可以形成具有鹅卵石外观的完整单层（图 1A-D）。</li>
	<li>将足量的 PBS、完全扩增培养基和无动物成分 （ACF） 细胞解离试剂盒预热至 37 °C。</li>
	<li>用 3 mL PBS 轻轻冲洗细胞。</li>
	<li>进行酶解离。<ol><li>向培养皿中加入 3 mL ACF 酶解离溶液，并在 37 °C 下孵育 5 分钟。移液后，轻轻去除细胞，并将细胞悬液转移到 3 mL ACF 酶抑制溶液中。</li>
		<li>重复加入 3 mL ACF 酶解离溶液，并再次孵育 5 分钟，以确保细胞分离程度最高。</li>
	</ol></li>
	<li>将细胞悬液在 400 x g 下在 4 °C 下旋转 5 分钟。 丢弃上清液。</li>
	<li>将细胞重悬于 1 mL 扩增培养基中。</li>
	<li>使用台盼蓝和血细胞计数器计数活细胞。</li>
	<li>将 P1 细胞悬液接种在几个预先涂有大鼠尾胶原的 100 mm 培养皿（5 x 104 个活细胞/cm2）上，并在 37 °C、5% CO2 的培养箱中培养细胞。</li>
</ol>3. 原代小鼠气道上皮细胞在气液界面的分化和 CSE 刺激
<ol><li>确认细胞类型。<ol><li>使用免疫荧光检测 （IFA） 和细胞角蛋白抗体验证分离细胞的上皮来源。将细胞接种在涂有大鼠尾胶原的载玻片上，并在细胞达到 80% 汇合后使用多聚甲醛将其固定至少 12 小时。</li>
		<li>用 PBS 溶液洗涤载玻片 3 次 5 分钟，并在室温 （RT） 下与 3% BSA 和 0.1% triton X-100 在 PBS 溶液中孵育 1 小时。</li>
		<li>将玻片在 4 °C 下与市售 1：500 稀释度的抗泛细胞角蛋白抗体孵育过夜。</li>
		<li>第二天，用 PBS 溶液洗涤玻片 3 次，每次 5 分钟，然后用稀释度为 1：1000 的二抗缓冲液在 RT 下在黑暗中孵育 2 小时。</li>
		<li>孵育后，用 PBS 洗涤玻片 3 次（每次洗涤 5 分钟），并向其中添加稀释度为 1：1000 的 DAPI。在 RT 孵育 15 分钟后，用 PBS 洗涤玻片一次 5 分钟。</li>
		<li>在荧光显微镜下观察载玻片，并将表达模式与阳性对照（A549 细胞系）和阴性对照（Raw264.7 细胞系）进行比较（图 2）。</li>
	</ol></li>
	<li>接种细胞以进行分化。<ol><li>如前所述分离并离心 P2 细胞，并将细胞沉淀重悬于适当体积的扩增培养基中，以便于接种 1 x 104 个活细胞/cm2。</li>
		<li>将 1 mL 细胞悬液移液到 transwell 聚碳酸酯膜插入物的顶腔上。向 transwell 的基底隔室中，添加 1.5 mL 增殖培养基。</li>
	</ol></li>
	<li>将细胞在 37 °C 下孵育，并使用扩增培养基每 3 天完全更换基底和顶端腔中的所有培养基，直至达到汇合。这通常需要 3-6 天。</li>
	<li>向 44.15 mL ALI 基础培养基中加入 5 mL ALI 10x 添加剂、500 μL ALI 维持添加剂、100 μL 肝素溶液和 250 μL 氢化可的松储备液，制备 50 mL 完全分化培养基。</li>
	<li>准备 CSE。<ol><li>将一支香烟通过 12.5 mL 分化培养基起泡，然后通过 0.22 μm 孔径过滤器过滤以制备 CSE。为确保 CSE 实验和批次之间的标准化，请在分光光度计上测量 320 nm 处的吸光度，并将 1 的光密度 （OD） 定义为 100%8。</li>
	</ol></li>
	<li>使用含有适当浓度 CSE 的分化培养基刺激原代小鼠气道上皮细胞，以检测对细胞分化的影响。</li>
	<li>让细胞分化 28 天，在此期间更换基底腔培养基，并通过去除顶腔培养基并用含有适当浓度 CSE 的分化培养基替换基底腔培养基，每周洗涤顶端侧两次。</li>
	<li>分析暴露后的细胞。<ol><li>暴露于含有 CSE 的分化培养基后，在任何时间点（即 0-28 天）收获细胞和培养上清液，以检测形态变化（即免疫荧光测定 （IFA） 或苏木精-伊红染色 （HE） 或用于生物信息学分析程序（即转录组学、蛋白质组学、代谢组学等）。</li>
	</ol></li>
</ol>

小鼠气道上皮细胞和慢性烟雾暴露的体外模型

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作者没有什么可披露的。

COPD 是一种常见的慢性气道炎症性疾病。暴露于烟草烟雾会导致慢性气道炎症、气道重塑和肺结构破坏，这是各种结构细胞和免疫细胞相互作用的结果10。气道上皮细胞作为肺部先天免疫系统的前线，在疾病的发展过程中起着非常重要的作用11。从这个角度来看，阐明上皮细胞在有害物质的刺激下如何改变和调节下游细胞，是一个亟待解决的科学问题。
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慢性阻塞性肺疾病 （COPD） 是一种具有复杂特征的异质性肺部疾病，而 COPD 患者逐渐趋于年轻化1。吸烟是COPD 2 的主要危险因素，对气道上皮细胞有深远影响，气道上皮细胞是抵御烟草烟雾的初始屏障。尽管存在这种已知的关联，但烟草烟雾诱导气道上皮细胞变化的详细机制仍未得到充分探索。彻底了解这些分子改变对于确定 COPD 的早期诊断标志物和治疗靶点至关重要。
为了解决这一差距，我们开发了一种使用小鼠气道上皮细胞的新型体外模型。这些细胞受到香烟烟雾提取物 （CSE） 的长期刺激，使我们能够监测动态细胞变化并探索气道上皮细胞在长期烟草刺激下的变化及其潜在机制。在该模型中，transwell 用于提供气道上皮细胞的气液界面，在上皮细胞分化的早期刺激香烟烟雾提取物，直到 28 天分化结束。以前的研究仅调查了气道上皮细胞的分化和短期刺激 （细胞系优势）。它们仅限于单一的调节途径 3,4,5,6。然而，这里介绍的方案使用原代小鼠气道上皮细胞，并优化了细胞提取过程，以获得比以前的气道上皮细胞培养方法更好的细胞活性。该模型侧重于细胞分化的改变以及全面的转录组学、蛋白质组学、代谢组学和表观基因组学分析。采用免疫荧光和先进的多组学技术，我们旨在阐明气道上皮细胞对慢性烟草烟雾暴露的细胞反应，从而有助于更深入地了解 COPD 发病机制。该模型可用于探究各种污染物长期刺激引起的气道上皮细胞分化模式的变化及其机制。

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			<td>24 mm Transwell with 0.4 &#181;m Pore Polyester Membrane Insert, Sterile</td>
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			<td>40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
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			<td>500x Gentamicin/Amphotericin Solution</td>
			<td>Gibco</td>
			<td>R01510</td>
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			<td>acetylated &#945;-Tubulin</td>
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			<td>#5335</td>
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			<td>Acetyl-&#945;-Tubulin (Lys40) (D20G3)XP Rabbit mAb&#160;</td>
			<td>cellsignal</td>
			<td>#5335</td>
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			<td>Animal Component Free Cell Dissociation Kit</td>
			<td>Stemcell</td>
			<td>05426</td>
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			<td>Anti-pan Cytokeratin antibody</td>
			<td>abcam</td>
			<td>ab7753</td>
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			<td>Cigarette</td>
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			<td>Claudin3</td>
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			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
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			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma</td>
			<td>DN25</td>
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			<td>Ham&#8217;s F-12</td>
			<td>Sigma-Aldrich</td>
			<td>N6658</td>
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			<td>Heparin Solution&#160;</td>
			<td>Stemcell</td>
			<td>07980</td>
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			<td>Hydrocortisone Stock Solution</td>
			<td>Stemcell</td>
			<td>07925</td>
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			<td>Mucin 5AC</td>
			<td>abcam</td>
			<td>ab212636</td>
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			<td>Occludin</td>
			<td>proteintech</td>
			<td>27260-1-AP</td>
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			<td>PBS</td>
			<td>Cytosci</td>
			<td>CBS004S-BR500</td>
			<td></td>
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			<td>Penicillin-Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15140122</td>
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			<td>PneumaCult-ALI 
			Basal Medium</td>
			<td>Stemcell</td>
			<td>05002&#160;</td>
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			<td>PneumaCult-ALI 10x Supplement</td>
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			<td>05003&#160;</td>
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			<td>PneumaCult-ALI Maintenance Supplement</td>
			<td>Stemcell</td>
			<td>05006</td>
			<td></td>
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			<td>PneumaCult-Ex Plus 50x Supplement</td>
			<td>Stemcell</td>
			<td>05042</td>
			<td></td>
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			<td>PneumaCult-Ex Plus Basal Medium</td>
			<td>Stemcell</td>
			<td>05041</td>
			<td></td>
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			<td>Pronase E</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
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			<td>Rat tail collagen</td>
			<td>Corning</td>
			<td>354236</td>
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			<td>Trypan Blue</td>
			<td>Stemcell</td>
			<td>07050&#160;</td>
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in vitro model for studying differentiation and changes of multi-omics on murine airway epithelial cells stimulated with cigarette smoke extract

慢性阻塞性肺病 （COPD） 主要归因于烟草烟雾暴露。研究气道上皮细胞如何在功能上适应烟草烟雾对于了解 COPD 的发病机制至关重要。本研究是使用原代小鼠气道上皮细胞建立一个 体外 模型，以模拟烟草烟雾的现实影响。与已建立的细胞系不同，原代细胞保留了更多的 体内样特性，包括生长模式、衰老和分化。这些细胞表现出敏感的炎症反应和有效的分化，因此与生理状况密切相关。在该模型中，原代小鼠气道上皮细胞在气液界面下以最佳浓度的香烟烟雾提取物 （CSE） 培养 28 天，这导致单层未分化细胞转化为假复层柱状上皮，表明 CSE 驯化。然后应用全面的多组学分析来阐明 CSE 影响基底气道细胞分化的机制。这些见解有助于更深入地了解支撑 COPD 进展以应对烟草烟雾暴露的细胞过程。

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition with complex characteristics, while patients with COPD gradually tend to be younger1. Smoking, a primary risk factor for COPD2, has a profound impact on airway epithelial cells, which serve as the initial barrier against tobacco smoke. Despite this known association, the detailed mechanisms through which tobacco smoke induces changes in airway epithelial cells remain inadequately explored. A thorough understanding of these molecular alterations is essential for identifying early diagnostic markers and therapeutic targets for COPD.
To address this gap, we developed a novel in vitro model using murine airway epithelial cells. These cells were subjected to long-term stimulation with cigarette smoke extract (CSE), enabling us to monitor dynamic cellular changes and explore the changes in airway epithelial cells under long-term tobacco stimulation and the underlying mechanism. In this model, transwell was used to provide the air-liquid interface of airway epithelial cells, and cigarette smoke extract was stimulated in the early stage of epithelial cell differentiation until the end of differentiation at 28 days. Previous studies investigated only the differentiation and short-term stimulation of airway epithelial cells (cell line dominance). They were limited to a single regulatory pathway3,4,5,6. However, the protocol presented here uses primary mouse airway epithelial cells and optimizes the cell extraction process for better cell activity than previous airway epithelial cell culture methods. This model focuses on alterations in cellular differentiation alongside comprehensive transcriptomic, proteomic, metabolomic, and epigenomic analyses. Employing immunofluorescence and advanced multi-omics techniques, we aimed to elucidate the cellular responses of airway epithelial cells to chronic tobacco smoke exposure, thereby contributing to a deeper understanding of COPD pathogenesis. This model can be used to explore the changes in airway epithelial cell differentiation pattern and its mechanism caused by long-term stimulation of various pollutants.

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

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

分化 小鼠气道上皮细胞在气液界面用分化培养基培养 28 天后成功分化。纤毛标志物乙酰化 α-微管蛋白（绿色; 图 3A）和杯状细胞标记物 Mucin5AC，分别为 6（红色; 图 3B）。
测定用于细胞分化的 CSE 浓度 用不同浓度的 CSE 刺激分离的上皮细胞 24 小时。结果表明，当 CSE 浓度高于 6% 时，上皮细胞的细胞活?...

在这里，我们描述了一种用于分离和分化小鼠气道上皮细胞的 体外 模型，重点关注它们对慢性香烟烟雾提取物 （CSE） 的适应。该模型可用于全面表征 CSE 的多组学影响，这可能为了解慢性烟雾暴露下的细胞反应提供见解。

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

in-vitro-model-for-studying-differentiation-changes-multi-omics-on

Research

JoVE Journal

Biology

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

516 次观看.  Capital Medical University. 我们的研究范围是使用气道上皮细胞开发和表征体外模型，以研究慢性 CSC 暴露的影响。我们旨在了解在长期香烟烟雾暴露下气道上皮细胞发生的细胞反应，这与 COPD 的发病机制有关。认识到 COPD 中错综复杂的上皮免疫细胞相互作用被认为是 COPD 研究中的关键最新进展。我们已经建立了一种新的体外模型，模拟气道上皮细胞中的慢性 CSC 暴露。?...

视频: 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

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.

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.