JoVE Journal > Immunology and Infection
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Abstract
Introduction
Protocol
Results
Discussion
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免疫与感染

marinum 分枝杆菌感染成人斑马鱼结核的建模

Published: October 08, 2018
doi:
10.3791/58299
, , ,
1Faculty of Medicine and Life Sciences,University of Tampere, 2Oral and Maxillofacial Unit,Tampere University Hospital

Summary

在这里, 我们提出了一个在成年斑马鱼使用其天然致病菌分枝杆菌 marinum的人结核病模型的协议。从被感染的斑马鱼内脏中提取的 DNA 和 RNA 可以用来揭示鱼的总结核杆菌负荷和宿主对 qPCR 的免疫应答。

Abstract

结核分枝杆菌是目前最致命的人类病原体, 每年造成170万人死亡和1040万次感染。接触这种细菌会引起人类广泛的疾病谱, 从消毒感染到积极进步的致命疾病不等。最常见的形式是潜在的肺结核, 这是无症状的, 但有可能重新激活为暴发性疾病。成年斑马鱼及其天然病原体marinum 分枝杆菌最近被证明是研究结核病广泛疾病谱的适用模型。重要的是, 该模型可以研究结核杆菌感染背景下的自发潜伏期和重新激活以及适应性免疫反应。本文介绍了成年斑马鱼实验性感染的方法, 收集了用于提取核酸的结核杆菌负荷测定和宿主免疫应答的体内器官。在内部开发的, m. marinum特异性 qPCR 检测比传统的电镀方法更敏感, 因为它也检测非分裂, 休眠或最近死的分枝杆菌的 DNA。由于 DNA 和 RNA 都是从同一个体中提取出来的, 因此可以研究患病状态与寄主和病原体基因表达之间的关系。因此, 成年斑马鱼的结核病模型作为一种高度适用的非哺乳动物体内系统来研究寄主-病原体的相互作用。

Introduction

斑马鱼 (斑马斑马) 是生物医学研究中广泛使用的动物模型, 是常见脊椎动物生物学的公认模型。斑马鱼已经适应了许多领域的研究模拟人类疾病和疾病, 从癌症1和心脏病2到感染和免疫学研究的几个细菌3和病毒感染4,5. 此外, 斑马鱼胚胎的前子宫发育使斑马鱼成为发育生物学6和毒理学7,8的流行模式。

在许多研究领域, 包括感染生物学, 通常使用光学透明斑马鱼幼虫。第一个免疫细胞出现在 24 h 后受精 (hpf), 当原始巨噬细胞被检测到9。中性粒细胞是下一个免疫细胞, 出现在 33 hpf10左右。因此, 斑马鱼幼虫是可行的研究早期感染阶段和先天免疫的作用, 在缺乏自适应免疫细胞11。然而, 成年斑马鱼具有完全功能的适应性免疫系统, 为感染实验提供了额外的复杂性层。T 细胞可以检测到3天后受精12, B 细胞能够产生功能性抗体4周后受精13。成年斑马鱼拥有哺乳动物先天和适应性免疫系统的所有主要对应物。在抗体 isotypes 和淋巴组织解剖中发现了鱼类和人类免疫系统的主要区别。斑马鱼只有三抗体类14, 而人类有五15。在缺乏骨髓和淋巴结的情况下, 鱼的主要淋巴器官是肾脏和胸腺16和脾脏、肾脏和肠道作为次要淋巴器官17。尽管有这些差异, 其完全免疫武库的先天和自适应细胞, 成年斑马鱼是一个高度适用, 易于使用, 非哺乳动物模型的寄主-病原体相互作用的研究。

斑马鱼最近被建立了作为一个可行的模型研究结核病18,19,20,21,22。肺结核是由结核分枝杆菌引起的一种机载疾病。根据世界卫生组织的数据, 结核病在2016年造成1.7人死亡, 是全球23个单一病原体的主要死因。老鼠24、25、兔子26和非人类灵长类27是结核病研究中最著名的动物模型, 但它们都面临着局限性。结核菌感染的非人类灵长类模型最接近人类疾病, 但由于严重的伦理考虑, 使用这种模式是有限的。其他动物模型受到 m. 型结核病宿主特异性的阻碍, 影响到疾病病理学。结核病建模的最大问题可能是人类疾病中广泛的感染和疾病结果: 结核病是一种非常异构的疾病, 从杀菌免疫到潜在的、活跃的和重新激活的感染28, 这可能很难重现和实验模型。

marinum 分枝杆菌是 m.结核的近亲, 有3000同源蛋白, 85% 的氨基酸标识29。m. marinum自然感染斑马鱼生产肉芽肿, 肺结核的标志, 在其内脏 19,30。与其他用于结核病研究的动物模型不同, 斑马鱼生产许多后代, 它只需要有限的空间, 重要的是, 它是 neurophysiologically 最不发达的脊椎动物结核模型。此外, m marinum感染导致潜伏感染, 积极的疾病, 甚至杀菌的结核杆菌感染的成年斑马鱼密切模仿疾病的结果, 人类结核病19,31,32. 在这里, 我们描述了成人斑马鱼的实验性结核模型的方法, 注射m. marinum入腹腔, 并利用定量 PCR 测量斑马鱼的结核杆菌负荷和免疫应答组织样本。

Protocol

所有斑马鱼实验都已获得芬兰动物实验委员会 (ESAVI/8245/04.10.07/2015) 的批准。方法是根据《法令》 (497/2013) 和政府关于保护在芬兰用于科学或教育目的的动物的法令 (564/2013) 执行的。 1. marinum 分枝杆菌的培养 注: 由于 marinum 分枝杆菌是一种能够引起人类表面感染的病原体, 在开始与这种细菌合作之前, 找出当地的人身安全和生物危害废物处置指南。 …

Representative Results

天然鱼类病原杆菌 marinum感染斑马鱼的内脏, 并产生全身性感染, 组织学可见肉芽肿19。成年斑马鱼通过腹腔注射感染m marinum 。用 dna 作为模板, 提取 dna 和 RNA, 用定量聚合酶链反应 (qPCR) 测定结核杆菌负荷。方法的轮廓如图 1所示。 最初用于感染鱼的分枝杆菌数量是感染结果?…

Discussion

这里我们描述了一个基于 qPCR 的应用来测量从实验感染的成年斑马鱼组织中提取的 DNA 结核杆菌负载。这个应用是基于引物设计围绕十六年代-二十三年代 rRNA 内部转录间隔序列40。用从已知数量的培养的分枝杆菌中提取的 DNA 制备的标准曲线估计鱼类样品中的总结核杆菌负荷, 并假设某一细菌在任何特定时刻都有一份其基因组副本。m. marinum-qPCR 的检测限为约100个菌落形成单…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到芬兰文化基金会、坦佩雷结核病基金会 (M.V.、M.M.H.、宪兵)、芬兰抗结核协会 (Suomen Tuberkuloosin Vastustamisyhdistyksen säätiö) 基金会的支持。M.M.H.、宪兵)、西格丽德 jusélius 基金会 (mp)、埃米尔 Aaltonen 基金会 (M.M.H.)、简和 Aatos Erkko 基金会 (mp) 和芬兰科学院 (mp)。琳娜 mäkinen, 汉娜-琳娜卡里和珍娜 ilomäki 被承认为他们的技术援助。作者承认坦佩雷斑马鱼实验室为他们的服务。

Materials

Mycobacterium marinum American Type Culture Collection ATCC 927
Middlebrock 7H10 agar BD, Thermo Fisher Scientific 11799042
Middlebrock OADC enrichment BD, Thermo Fisher Scientific 11718173
Middlebrock 7H9 medium BD, Thermo Fisher Scientific 11753473
Middlebrock ADC enrichment BD, Thermo Fisher Scientific 11718173
Tween 80 Sigma-Aldrich P1754
Glycerol Sigma-Aldrich G5516-500ML
GENESYS20 Spectrophotometer Thermo Fisher Scientific
Phosphate buffered saline tablets (PBS) Sigma-Aldrich P4417-50TAB
Phenol red Sigma-Aldrich P3532
27G needle Henke Sass Wolf 4710004020
1 ml syringe Henke Sass Wolf 4010.200V0
Omnican 100 30G insulin needle Braun 9151133
3-aminobenzoic acid ethyl ester (pH 7.0) Sigma-Aldrich A5040
1.5 ml homogenization tube Qiagen 13119-1000
2.8 mm ceramic beads Qiagen 13114-325
Ethanol, ETAX Aa Altia
2-propanol Sigma-Aldrich 278475
Chloroform VWR 22711.290
Guanidine thiocyanate Sigma-Aldrich G9277 FW 118.2 g/mol
Sodium citrate Sigma-Aldrich 1613859 FW 294.1 g/mol
Tris (free base) Sigma-Aldrich TRIS-RO FW 121.14 g/mol
TRI reagent Molecular Research Center TR118 Guanidine thiocyanate-phenol solution
PowerLyzer24 homogenizator Qiagen
Sonicator m08 Finnsonic
Nanodrop 2000 Thermo Fisher Scientific
SENSIFAST No-ROX SYBR, Green Master Mix Bioline BIO-98005
qPCR 96-well plate BioRad HSP9601
Optically transparent film BioRad MSB1001
C1000 Thermal cycler with CFX96 real-time system BioRad
RNase AWAY Thermo Fisher Scientific 10666421 decontamination reagent eliminating RNases
DNase I Thermo Fisher Scientific EN0525
Reverse Transcription Master Mix Fluidigm 100-6298
SsoFast Eva Green master mix BioRad 172-5211
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Tags

Keywords: Mycobacterium MarinumTuberculosisZebrafishAdaptive Immune ResponsesPathogenesisNucleic AcidBacterial LoadsGene Expression7H10 Plate7H9 MediumADC EnrichmentPolysorbate 80GlycerolCell Culture FlaskInoculation LoopOD600PBSPhenol Red27 Gauge Needle30 Gauge Insulin NeedleRag Mutant7H10 Plates
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Luukinen, H., Hammarén, M. M., Vanha-aho, L., Parikka, M. Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish. J. Vis. Exp. (140), e58299, doi:10.3791/58299 (2018).
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