Name: infection of primary nasal epithelial cells grown at an air-liquid interface to characterize human coronavirus-host interactions
Uploaded: 2023-09-22T13:20:00
Description: Watch this Scientific Journal Video about Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions at JoVE.com

本研究的资金来源如下：美国国立卫生研究院 （NIH） R01AI 169537（SRW 和 N.A.C.）、NIH R01AI 140442 （S.R.W.）、VA Merit Review CX001717 （N.A.C.）、VA Merit Review BX005432（S.R.W. 和 N.A.C.）、宾夕法尼亚大学冠状病毒和其他新兴病原体研究中心 （S.R.W.）、Laffey-McHugh 基金会（S.R.W. 和 N.A.C.）， T32 AI055400 （CJO）、T32 AI007324 （AF）。

鼻腔标本的使用得到了宾夕法尼亚大学机构审查委员会（协议#800614）和费城弗吉尼亚州机构审查委员会（协议#00781）的批准。
1.鼻腔ALI培养物感染
注：临床标本的采集以及鼻腔ALI培养物的生长和分化不在本文的讨论范围之内。培养原代鼻上皮细胞的具体方法可以在最近发表的利用这些培养物的著作中找到 18,22,23。<sup...

<ol>
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这里详述的方法描述了一种原代上皮培养系统，其中患者来源的鼻上皮细胞在气液界面上生长，并应用于HCoV-宿主相互作用的研究。一旦分化，这些鼻 ALI 培养物概括了 体内 鼻上皮的许多特征，包括具有纤毛细胞、杯状细胞和基底细胞的异质性细胞群，以及完整的粘液纤毛功能，纤毛和粘液分泌物跳动强劲。与传统的呼吸道上皮细胞系相比，这种异质细胞群是该培养系统的一个关键优势，?...

迄今为止，已发现七种人类冠状病毒 （HCoV），并引起一系列呼吸道疾病2。常见或季节性 HCoV（HCoV-NL63、-229E、-OC43 和 -HKU1）通常与上呼吸道病变相关，每年估计引起 10%-30% 的普通感冒病例。虽然这是与常见 HCoV 相关的典型临床表型，但这些病毒可在高危人群中引起更严重的下呼吸道疾病，包括儿童、老年人和免疫功能低下个体 3,4。在过去20年中，出现了三种致病性HCoV并造成了重大突发公共卫生事件，包括严重急性呼吸系统综合症（SARS）-CoV、中东呼吸系统综合症（MERS）-CoV和SARS-CoV-2。致命的HCoV与更严重的呼吸道病变有关，中东呼吸综合征冠状病毒病例的病死率为>34%（自2012年出现以来，2500多例病例中有894例死亡）5,6清楚地说明了这一点。值得注意的是，致命的HCoV还会引起一系列呼吸道疾病，从无症状感染到致命性肺炎，正如正在进行的COVID-19大流行所见7。
与其他呼吸道病原体一样，HCoV 进入呼吸道并在鼻上皮中建立生产性感染8。扩散到下气道被认为与从口腔/鼻腔到肺部的误吸有关，其中 HCoV 引起更显着的下呼吸道病变 9,10,11。因此，鼻子是病毒进入的初始门户，并且是感染的主要屏障，其强大的粘液纤毛清除机制和独特的先天免疫机制旨在防止病毒进一步传播到下气道12,13。例如，据报道，鼻上皮细胞表达高于平均水平的抗病毒干扰素和干扰素刺激基因的基础水平，这表明鼻细胞可能为对呼吸道病毒的早期反应做好准备14,15,16。
我们之前曾利用在气液界面 （ALI） 生长的患者来源的原代鼻上皮细胞来模拟 HCoV 感染开始的鼻子中的 HCoV 与宿主的相互作用。鼻腔 ALI 培养物对致病性（SARS-CoV-2 和 MERS-CoV）和常见 HCoV（HCoV-NL63 和 HCoV-229E）均有效，并且与传统的气道上皮细胞系（如 A549（一种肺腺癌细胞系））相比具有各种优势16,17。分化后，鼻ALI培养物包含异质性细胞群，并表现出体内鼻上皮预期的许多功能，例如粘膜纤毛清除机制18。与下气道培养系统（如人支气管上皮细胞，HBEC）相比，鼻细胞也具有优势，因为与使用支气管镜检查等技术获得HBECs相比，通过细胞学刷取鼻上皮细胞的侵入性要小得多19,20,21。
本文描述了利用这种鼻ALI培养系统来表征鼻上皮中HCoV-宿主相互作用的方法。我们在最近发表的作品中应用了这些方法来比较 SARS-CoV-2、MERS-CoV、HCoV-NL63 和 HCoV-229E1、16、17。尽管这些方法和代表性结果强调了该鼻细胞模型中HCoV的研究，但该系统对其他HCoV以及其他呼吸道病原体具有高度适应性。此外，这些方法可以更广泛地应用于其他ALI培养系统，以研究病毒复制和细胞嗜性，以及感染后的细胞毒性和先天免疫诱导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor secondary antibodies (488, 594, 647)</td>
			<td>Invitrogen</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (bovine serum albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete mini EDTA-free protease inhibitor</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotoxicity detection kit</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Media)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (Dulbecco&#39;s Phosphate Buffered Saline)</td>
			<td>Gibco</td>
			<td>14190136</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS + calcium + magnesium</td>
			<td>Gibco</td>
			<td>14040-117</td>
			<td></td>
		</tr>
		<tr>
			<td>Endohm-6G measurement chamber</td>
			<td>World Precision Instruments</td>
			<td>ENDOHM-6G</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial cell adhesion marker (EpCAM; CD326)</td>
			<td>eBiosciences</td>
			<td>14-9326-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial Volt/Ohm (TEER) Meter (EVOM)</td>
			<td>World Precision Instruments</td>
			<td>300523</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>HyClone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>FV10-ASW software for imaging</td>
			<td>Olympus</td>
			<td>Version 4.02</td>
			<td></td>
		</tr>
		<tr>
			<td>HCoV-NL63 (Human coronavirus, NL63)</td>
			<td>BEI Resources</td>
			<td>NR-470</td>
			<td></td>
		</tr>
		<tr>
			<td>HCoV-NL63 nucleocapsid antibody</td>
			<td>Sino Biological</td>
			<td>40641-V07E</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht stain</td>
			<td>Thermo Fisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BIO-RAD</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>LLC-MK2 cells</td>
			<td>ATCC</td>
			<td>CCL-7</td>
			<td>To titrate HCoV-NL63</td>
		</tr>
		<tr>
			<td>MERS-CoV (Human coronavirus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), EMC/2012)</td>
			<td>BEI Resources&#160;</td>
			<td>NR-44260</td>
			<td></td>
		</tr>
		<tr>
			<td>MERS-CoV nucleocapsid antibody</td>
			<td>Sino Biological</td>
			<td>40068-MM10</td>
			<td></td>
		</tr>
		<tr>
			<td>MUC5AC antibody</td>
			<td>Sigma-Aldrich</td>
			<td>AMAB91539</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus Fluoview confocal microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 647 stain</td>
			<td>Abcam</td>
			<td>ab176759</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosStop easy pack (phosphatase inhibitors)&#160;</td>
			<td>Roche</td>
			<td>PHOSS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader&#160;</td>
			<td>Perkin Elmer</td>
			<td>HH34000000</td>
			<td>Any plate reader or ELISA reader is sufficient; must be able to read absorbance at 492 nm</td>
		</tr>
		<tr>
			<td>RIPA buffer (50 mM Tris pH 8; 150 mM NaCl; 0.5% deoxycholate; 0.1% SDS; 1% NP40)</td>
			<td>Thermo Fisher</td>
			<td>89990</td>
			<td>Can prep in-house or purchase</td>
		</tr>
		<tr>
			<td>RNeasy Plus Kit</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 (SARS-Related Coronavirus 2, Isolate USA-WA1/2020)</td>
			<td>BEI Resources</td>
			<td>NR-52281</td>
			<td></td>
		</tr>
		<tr>
			<td>SARS-CoV-2 nucleocapsid antibody</td>
			<td>Genetex</td>
			<td>GTX135357</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Fisher Scientific</td>
			<td>BP151100</td>
			<td></td>
		</tr>
		<tr>
			<td>Type IV &#946;- tubulin antibody</td>
			<td>Abcam</td>
			<td>ab11315</td>
			<td></td>
		</tr>
		<tr>
			<td>VeroCCL81 cells</td>
			<td>ATCC</td>
			<td>CCL-81</td>
			<td>To titrate MERS-CoV</td>
		</tr>
		<tr>
			<td>VeroE6 cells</td>
			<td>ATCC</td>
			<td>CRL-1586</td>
			<td>To titrate SARS-CoV-2</td>
		</tr>
	</tbody>
</table>

infection of primary nasal epithelial cells grown at an air-liquid interface to characterize human coronavirus-host interactions

在过去20年中，三种高致病性人类冠状病毒（HCoV）——SARS-CoV（2002年）、MERS-CoV（2012年）和SARS-CoV-2（2019年）——已经出现并引发了重大的公共卫生危机。另外四种HCoV每年导致很大一部分普通感冒病例（HCoV-NL63、-229E、-OC43和-HKU1），这凸显了在生理相关系统中研究这些病毒的重要性。HCoV进入呼吸道并在鼻上皮中建立感染，鼻上皮是所有呼吸道病原体遇到的主要部位。我们使用原代鼻上皮培养系统，其中患者来源的鼻腔样本在气液界面 （ALI） 生长，以研究这个重要前哨位点的宿主-病原体相互作用。这些培养物概括了 体内 气道的许多特征，包括存在的细胞类型、纤毛功能和粘液产生。我们描述了表征 HCoV 感染后鼻腔 ALI 培养物中病毒复制、宿主细胞嗜性、病毒诱导的细胞毒性和先天免疫诱导的方法，以最近比较致死性和季节性 HCoV 的工作为例1。对鼻子中宿主-病原体相互作用的了解的增加有可能为针对HCoV和其他呼吸道病毒的抗病毒疗法提供新的靶点，这些病毒可能会在未来出现。

代表性数字部分改编自手稿 Otter 等人 1 中的数据。根据上述方案，来自四个或六个供体的鼻腔 ALI 培养物感染了四种 HCoV（SARS-CoV-2、MERS-CoV、HCoV-NL63 和 HCoV-229E）中的一种，每种病毒的平均顶端脱落病毒滴度如图 1A 所示。虽然所有这四种 HCoV 在鼻腔 ALI 培养物中都能有效复制，但 SARS-CoV-2 和 HCoV-229E 的复制效率最高。请注意，这些是平均病毒滴度，并且最近?...

Watch this Scientific Journal Video about Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions at JoVE.com

Infection of Primary Nasal Epithelial Cells Grown at an Air-Liquid Interface to Characterize Human Coronavirus-Host Interactions

鼻上皮是所有呼吸道病原体遇到的主要屏障部位。在这里，我们概述了使用作为气液界面 （ALI） 培养物生长的原代鼻上皮细胞来表征生理相关系统中人类冠状病毒与宿主相互作用的方法。

在气液界面上生长的原代鼻上皮细胞的感染，以表征人类冠状病毒与宿主的相互作用

Three highly pathogenic human coronaviruses (HCoVs) - SARS-CoV (2002), MERS-CoV (2012), and SARS-CoV-2 (2019) - have emerged and caused significant public ...

Research

JoVE Journal

Immunology and Infection

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

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 ...

Following Cell-fate in E. coli After Infection by Phage Lambda

Following Cell-fate in E. coli After Infection by Phage Lambda

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Biomimetic Materials to Characterize Bacteria-host Interactions

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

人胎盘及蜕膜器官培养研究在母胎界面感染

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...