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Immunologia e infezioni

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Published: April 20, 2021
doi:
10.3791/62314
, ,
1Department of Developmental Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology,Ministry of Education, China Medical University

Summary

大肠杆菌 是新生儿革兰阴性细菌性脑膜炎的主要原因。在细菌感染期间，中性粒细胞产生的活性氧物种起着主要的杀菌作用。在这里，我们介绍了一种检测中性粒细胞中反应性氧物种以应对 大肠杆菌脑膜炎的方法。

Abstract

大肠杆菌 （大肠杆菌）是导致新生儿脑膜炎的最常见革兰阴性细菌。细菌血症的发生和细菌通过血脑屏障的渗透是 大肠杆菌 脑膜炎发展的不可或缺的步骤。活性氧物种（ROS）是中性粒细胞破坏入侵病原体的主要杀菌机制。在本协议中，使用实时荧光微板读卡器检测到的荧光 ROS 探头对感染脑膜 大肠杆菌 的嗜中性粒细胞内 ROS 生产的时间依赖性 ROS 进行量化。该方法还可用于在病原体-宿主相互作用期间评估哺乳动物细胞中的ROS生产。

Introduction

新生儿细菌性脑膜炎是一种常见的儿科传染病。大肠杆菌（大肠杆菌）与K1胶囊是最常见的革兰阴性病原体引起新生儿细菌性脑膜炎，约占总发病率的80%1，2，3。尽管抗菌化疗和支持性护理取得了进展，但细菌性脑膜炎仍然是发病率高、死亡率最高的疾病之一。

新生儿细菌性脑膜炎的发生通常从致病菌从新生儿的局部病变进入周围循环引起的细菌血症开始，然后通过血脑屏障（BBB）渗透到大脑中，导致脑膜炎。细菌血症的发病取决于细菌与宿主免疫细胞（包括嗜中性粒细胞和巨噬细胞等）之间的相互作用。中性粒细胞占白细胞的50-70%，是抵御细菌感染的第一道防线。在细菌入侵期间，活性嗜中性粒细胞被招募到传染性部位并释放活性氧物种（ROS），包括超氧化物离子、过氧化氢、羟基基和单氧7。ROS对细菌的细胞膜、核酸分子和蛋白质进行氧化反应，导致入侵细菌8的损伤和死亡。线粒体是俄罗斯在真核细胞中生产的主要场所，各种氧化物（如烟酰胺腺苷二氯酰胺磷酸盐（NADPH）氧化酶复合物、脂氧酶系统、蛋白激酶C和环氧酶酶系统）调解了ROS9、10的生产。代表中性粒细胞中主要抗菌机制的ROS生产的实时测量，是研究细菌与宿主相互作用期间宿主防御的有用方法。

在此协议中，受脑膜性大肠杆菌 感染的嗜中性粒细胞中依赖时间的 ROS 生产用荧光 ROS 探头 DHE 进行量化，该探测器由实时荧光微板读卡器检测到。在病原体与宿主相互作用期间，此方法还可用于评估其他哺乳动物细胞中的 ROS 生产。

Protocol

本研究中应用的志愿者外周血液经中国医科大学第一医院机构评审委员会批准（#2020-2020-237-2）。 1. 试剂和文化介质的制备 通过添加 8.29 克 NH4Cl、1 克 KHCO 3、37.2 毫克 Na2EDTA 添加到 1 升双蒸馏水中，将 pH 值调整为 7.2-7.4，从而准备红细胞裂解缓冲器。使用 0.22 μm 过滤器过滤细菌。 通过在RPMI 1640介质中加入5%的胎儿牛血清，并在4°C储存，…

Representative Results

使用本文概述的协议，嗜中性粒细胞从人类外周血液中分离出来，并装载荧光探头 DHE，以检测针对 E44 感染的 ROS 水平的变化。在这里，我们提供具有代表性的数据，演示由微板读卡器实时确定的 E44 菌株引起的 ROS 生产。通过在 100 的 MOI 中添加 E44 菌株，ROS 水平立即增加，并以时间依赖的方式呈现持续上升趋势（图 1）。通过添加PMA，一个众所周知的中性粒体细胞内ROS的ROS?…

Discussion

嗜中性粒细胞是人类血液循环中白血球最丰富的成分。它们是先天人类免疫系统中的重要效应细胞，是抵御病原体入侵的第一道防线。ROS的产生代表了11号噬菌体病后嗜中性粒细胞的主要杀菌机制之一。最近的研究表明，一种名为中性粒细胞外陷阱（NET）的中性粒细胞释放出的网状结构也参与了细菌杀灭过程6、11、12。</su…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了中国国家自然科学基金委员会（31670845、31870832、32000811）和辽宁省杰出教授项目（LJH2018-35）的支持。

Materials

15 mL polypropylene conical centrifuge tubes KIRGEN KG2611
96-well plate Corning 3025
Agar DINGGUO DH010-1.1
Autuomated cell counter Bio-rad 508BR03397
Biological Safety Carbinet Shanghai Lishen Hfsafe-1200Lcb2
Brain heart infusion BD 237500
CD16 Microbeads, human Miltenyi Biotec 130-045-701
Centrifuge Changsha Xiangyi TDZ5-WS
Columns Miltenyi Biotec 130-042-401
Dihydroethidium (DHE) MedChemExpress 104821-25-2
Fetal bovine serum Cellmax SA211.02
Incubator Heraeus Hera Cell
MACS separation buffer Miltenyi Biotec 130-091-221
Microplate Reader Molecular Devices SpectraMax M5
Phorbol 12-myristate 13-acetate (PMA) Beyoitme S1819-1mg
QuadroMACS separation Unit Miltenyi Biotec 130-090-976
Rifampicin Solarbio 13292-46-1
RPMI1640 medium Sangon Biotech E600027-0500
Thermostatic shaker Shanghai Zhicheng ZWY-100D
Trypton OXOID LP0042
Yeast extract OXOID LP0021
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Riferimenti

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Tags

Keywords: Reactive Oxygen SpeciesNeutrophilsEscherichia ColiMeningitisNeonatal Bacterial MeningitisBacteremiaBlood-brain BarrierHost DefenseROS ProductionBacterial ColonyBrain-Heart Infusion MediumRifampicinPeripheral Blood SamplesRed Blood Cell LysisMagnetic Cell SortingWhite Blood CellsNeutrophils
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Citazione di questo articolo
Zhang, X., An, M., Zhao, W. Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli. J. Vis. Exp. (170), e62314, doi:10.3791/62314 (2021).
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