JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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免疫与感染

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Published: January 06, 2014
doi:
10.3791/50823
, , ,
1Department of Pediatrics,Harvard Medical School, 2Department of Pediatric Gastroenterology,MGH for Children, 3Mucosal Immunology and Biology Research Center,Massachusetts General Hospital

Summary

中性粒细胞跨上皮迁移，以应对粘膜细菌感染，有助于上皮损伤和临床疾病。已经开发出一种 体外 模型，结合了病原体、人类嗜中性粒细胞和在转井过滤器上生长的极化人类上皮细胞层，以促进研究，以解开协调这一现象的分子机制。

Abstract

粘性表面是预防致病生物的防护屏障。当感应病原体导致组织与迁移的炎症细胞（主要是嗜中性粒细胞）的渗透时，先天免疫反应被激活。如果过度或处于未解决状态，此过程可能会对组织造成破坏性破坏。 可利用 体外 培养模型研究病原体诱导中性粒细胞跨上皮迁移中涉及的独特分子机制。这种类型的模型在实验设计中提供了多功能性，为控制病原体、上皮屏障或中性粒细胞提供了机会。在透水转井过滤器上生长的极性上皮单层的气质表面的致病感染，在生理上与玄武岩相关，对应用于玄武岩表面的嗜中性嗜血杆菌进行肛盖跨上皮迁移。此处描述的 体外 模型演示了在极化肺上皮单层中演示中性粒细胞迁移所需的多个步骤，该单层已感染致病性 P. aeruginosa （PAO1）。描述了用人类衍生的肺上皮细胞对透水转井进行播种和培养，同时从整个人类血液中分离中性粒细胞，并培养PAO1和非致病性K12 大肠杆菌 （MC1000）。 移民过程和对成功迁移的嗜中性粒细胞的定量分析，已动员起来应对致病性感染，并显示具有代表性的数据，包括正对照和负对照。这种 体外 模型系统可以操纵并应用于其他粘性表面。涉及中性粒细胞过度渗透的炎症反应可能对宿主组织具有破坏性，并且可能发生在没有致病性感染的情况下。通过实验操纵本文所述的 体外 共生检测系统，更好地了解促进中性粒细胞跨上皮迁移的分子机制，对于确定一系列粘膜传染病和炎症性疾病的新治疗目标具有重大潜力。

Introduction

粘胶表面作为物理和免疫屏障，为防止外部威胁在环境中普遍存在提供保护。当致病生物入侵2时，这种保护性上皮屏障可能会受到损害。在细菌病原体的情况下，这种相遇往往通过激活先天免疫系统和触发被称为嗜中性粒细胞2-4的第一反应器颗粒细胞的快速动员煽动炎症过程。促进中性粒细胞招募的化学诱因剂部分由粘膜上皮细胞产生，试图摆脱宿主的违规病原体2-4。粘膜上皮表面过度或未解决的中性粒细胞渗透可引起重大病理学1，5。这是抗菌中性粒细胞库5-7造成的非特异性组织损伤的结果。在这种情况下，中性粒细胞的细菌清除能力因传染性侮辱期间宿主组织的破坏而黯然失色。 保护性上皮屏障功能的破坏可导致基础组织对微生物和/或毒素的进一步暴露，进一步加剧疾病病理学8，9。这些后果可以在多个器官系统中观察到，包括肺和消化道1，5。此外，非感染性炎症条件，如严重的哮喘发作，慢性阻塞性肺病（COPD），急性呼吸窘迫综合征（ARDS）和炎症性肠病（IBD）的特点是，通过过度的嗜中性反应4，5，10-12的粘膜上皮屏障的病理突破。

粘体感染后中性粒细胞招募的复杂过程涉及几个分门别化步骤1，5，13，14。首先，嗜中性粒细胞必须通过一系列细胞对细胞的相互作用来脱离循环，促进跨内皮迁移1，13。中性粒细胞接下来导航现有的间歇空间，其中包含细胞外基质1，14。要到达受感染的粘膜的流明，中性粒细胞必须迁移穿过上皮屏障1，4，5。这种复杂的多步骤现象往往被综合使用体内动物模型感染15。这些模型有助于确定特定因素的必要性，如化疗因子、粘附分子或参与整个过程的信号通路，但在很大程度上不足以解决对每个不同分门别析步骤16至关重要的分子贡献。培养体外系统建模跨内皮，转矩阵，或中性粒细胞的跨上皮迁移在这方面特别有用1，14，16，17。

已发展出一套强大的共生检测系统，以破译负责中性粒细胞跨上皮迁移的机制，以应对18-22年的致病性感染。该模型涉及用细菌病原体感染极化人类上皮细胞层的 apical 表面，然后将新分离的人类中微子应用到基底层表面 18-22。中性粒细胞迁移跨越上皮屏障，以响应18，21-23年致病性感染后分泌的上皮衍生化学产品。该系统采用肠道和肺上皮培养物，暴露于适当的组织特定细菌病原体，并揭示了新的分子机制可能重要的中性粒细胞招募过程中，粘膜感染3，8，19，24-28。这种 体外 共生模式的优点是，一种还原方法使研究者能够在控制良好、可重现、相当便宜的系统中实验性地操纵病原体、上皮屏障和/或中性粒细胞。从这种方法中收集的见解可以有效地利用在中性粒细胞招募期间使用 体内 感染模型22，29，30进行分门别析事件的集中分析。

本文演示了成功建立这种可重复模型以探索病原体诱发的中微子跨上皮迁移所需的多个步骤。本文介绍了感染病原体 伪多莫纳斯动脉鲁吉诺萨的 肺上皮屏障：然而，其他组织表皮和病原体可以替代与轻微的修改。在倒置胶原蛋白涂层透气透水过滤器上对极化肺上皮细胞层的播种和培养进行了详细的介绍，致病性 P.aeruginosa 的生长和中微粒细胞从全血中分离也详细介绍了这一点。这些组件如何结合观察病原体诱发的中微子跨上皮迁移，并结合适当的正负对照组进行，以建立可重复的检测。本文结合文献中的具体研究，探讨了这种方法研究病原体诱导中性粒细胞跨上皮迁移各个方面的多功能性。

Protocol

步骤 （1-3） 应在层压流罩下的无菌环境中执行。 1. 胶原蛋白涂层透水井 制作 30μg/ml 胶原蛋白溶液。稀释 3 毫克/毫升胶原蛋白库存 1：100 在 60% 乙醇，已通过 0.2 μm 过滤器单元。 漩涡稀释了 30μg/ml 溶液。注意：没有必要涡流3毫克/毫升胶原蛋白库存溶液，因为这个解决方案是相当粘稠的，气泡可能会引入，可能会降低管道时的准确性。 从外部?…

Representative Results

多项研究表明，病原体感染的上皮层促进中微子跨上皮迁移3，8，19，24-28，31，32。这发生在一个上皮细胞衍生的中性粒细胞化学梯度3，23的病原体特异性诱导。例如，致病 性P.aeruginosa 与肺上皮细胞的顶层相互作用，导致大量嗜中性粒细胞在上皮层18，22，25，26，33，34迁移。这种临床相关的检测系统可以以多种方式进行操作，在与适当的控制配对时，揭开关键病原?…

Discussion

中微霉素在粘膜上皮表面的迁移是感染细菌病原体后疾病病理学的一个常见特征。本文描述的方法提供了一种快速、直接的方法，利用人类细胞衍生的体外人工造影检测系统，对细菌感染引发的炎症过程的一个特征进行实验隔离。该系统最初是利用极化肠道上皮细胞开发的，该细胞感染了肠道病原体，包括沙门氏菌、西盖拉弹性内膜和各种致病性大肠杆菌8?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了NIH（1 R01 AI095338-01A1）的财政支持。

Materials

NCl-H292 cells ATCC CRL-1848
RPMI-1640 medium ATCC 30-2001
Pseudomonas aeruginosa PAO1 ATCC #47085
Escherichia coli MC1000 ATCC #39531
D-PBS (1x) liquid Invitrogen 14190-144 without calcium and magnesium
Heat Inactivated Fetal bovine serum Invitrogen 10082-147 10% added to culture medium
Penicillin-Streptomycin Invitrogen 15140-122 100x: 10,000 units of penicillin and 10,000 µg of streptomycin per ml.
Trypsin-EDTA (0.05%) Invitrogen 25300-062 50 ml aliquots are stored frozen at -20 ºC.  Aliquot in use can be stored at 4 ºC short-term.  
Hank's Balanced Salt Solution – HBSS(-) Invitrogen 14175-079 Sterile, without calcium and magnesium
Trypan Blue Solution Invitrogen 15250-061. Stock = 0.4%
Collagen, Rat Tail Invitrogen A10483-01 Can also be isolated in the laboratory directly from the tails of rats using standard protocols
Citric acid Sigma-Aldrich  C1909-500G Component of 1 M citrate buffer and acid citrate dextrose (ACD) solution
Sodium Citrate Sigma-Aldrich  S4641-500G Component of 1 M citrate buffer
Dextrose anhydrous Sigma-Aldrich  D8066-250G Component of acid citrate dextrose (ACD) solution
Ammonium Chloride Sigma-Aldrich  213330-500G Component of red blood cell (RBC) lysis buffer
Sodium bicarbonate Sigma-Aldrich  S6014-500G Component of red blood cell (RBC) lysis buffer
EDTA Sigma-Aldrich  ED-100G Component of red blood cell (RBC) lysis buffer
HBSS(+) powder Sigma-Aldrich  H1387-10L Key component of HBSS+
HEPES Sigma-Aldrich  H3375-500G Component of HBSS+
Sigmacote Sigma-Aldrich  SL2-25ML Follow vendor instructions to coat glass pipette tips
Triton X-100 Sigma-Aldrich  T-9284
2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) Sigma-Aldrich  A9941-50TAB Key component of ABTS substrate solution
30% Hydrogen Peroxide Solution Sigma-Aldrich  H1009-100ML Component of ABTS substrate solution
N-Formyl-Met-Leu-Phe (fMLP or fMLF) Sigma-Aldrich  F-3506 A Stock solution of 10 mM in DMSO should be prepared and aliquots stored at -20 ºC.
Gelatin Type B Fisher Scientific M-12026
Pseudomonas isolation agar  Fisher Scientific DF0927-17-1 Follow manufacturer’s instructions to make PIA plates
Ficoll-Paque PLUS  Fisher Scientific 45-001-749 Optional, can improve neutrophil purity
Name of Material / Equipment Company Catalog Number Comments
24-well migration plate Corning Incorporated #3524
24-well wash plate Falcon 35-1147 Can be reused if soaked in 70% ethanol and washed thoroughly prior to reuse
96-well plate Fisher Scientific #12565501
Transwell Permeable Supports  Corning Incorporated #3415 Polycarbonate; Diameter: 6.5 mm; Growth area: 0.33 cm2; Dish style: 24-well plate; Pore size: 3.0 µm
Petri dish Falcon 35-1013 Each Petri dish holds 24 inverted 0.33 cm2 Transwells.  
500 ml 0.2 μm filter / flask Fisher Scientific 09-740-25A To sterilize acid citrate dextrose (ACD) solution
5-3/4 in glass Pasteur pipette Fisher Scientific 13-678-20A Coat tips with Sigmacote prior to use
Hemostat Fisher Scientific 13-812-14 Curved, Serrated
Invertoskop Inverted Microscope Zeiss #342222
Versa-Max Microplate Reader Molecular Devices #432789
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Tags

Keywords: In Vitro CocultureNeutrophil Trans-epithelial MigrationMucosal ImmunityPathogenic InfectionPolarized Epithelial MonolayerNeutrophil InfiltrationInflammatory ResponseTherapeutic Targets
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Kusek, M. E., Pazos, M. A., Pirzai, W., Hurley, B. P. In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration. J. Vis. Exp. (83), e50823, doi:10.3791/50823 (2014).
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