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Immunologia e infezioni

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Published: July 30, 2012
doi:
10.3791/4051
,
1Department of Molecular and Biomedical Sciences,University of Maine

Summary

斑马鱼的快速发展，小尺寸和透明度是先天感染的免疫控制的研究的巨大优势<sup> 1-4</sup>。在这里，我们证明斑马鱼幼虫病原真菌感染的技术<em>白念珠菌</em>通过显微注射方法最近使用的牵连吞噬细胞NADPH氧化酶活性真菌异形控制<sup> 5</sup>。

Abstract

由病原体白念珠菌引起的播散性念珠菌是临床上重要的问题，在住院的个人和30到40％的占死亡率6。系统性念珠菌通常是由先天免疫控制，并与先天免疫细胞成分如巨噬细胞NADPH氧化酶的遗传缺陷的人更易患念珠菌7-9。很少有人知道关于C 的动态白念珠菌与先天免疫细胞在体内的相互作用。在体外研究中广泛建立了外部主机C.白念珠菌的巨噬细胞内发芽，迅速 ​​摧毁了嗜中性粒细胞10-14。 在体外研究中，虽然有用，能不能概括在复杂的体内环境，细胞因子水平，细胞外基质的附件，以及10间的接触，其中包括随时间变化的动态， 15-18 </sup>。为了探讨这些因素在宿主 – 病原体相互作用的贡献，关键是找到一个模式生物，在现场的完整主机可视化的非侵入性感染的这些方面。

斑马鱼幼虫提供了一个独特的和多功能的脊椎动物宿主感染的研究。发展的斑马鱼幼虫的前30天，有先天免疫防御2，19-21，简化，如播散性念珠菌是高度依赖先天免疫疾病的研究。小斑马鱼幼虫的大小和透明度，使在细胞水平上的主机和病原体感染的动态影像。与先天免疫细胞荧光的转基因幼虫可用于识别特定的细胞类型涉及在感染22-24。修饰的反义寡核苷酸（吗啉代）可以用来击倒各种免疫成分，如巨噬细胞NADPH氧化酶研究在回应funga的变化L型感染5。除了使用小低脊椎动物的道德和实用性强等优点，斑马鱼幼虫提供了独特的图像之间的病原体和宿主在intravitally颜色的酣战的可能性。

斑马鱼已被用于模型感染人类致病细菌的数量，并一直在我们理解分枝杆菌感染，25器乐重大进展。然而，最近才具有更大的病原体，如被用于真菌感染的幼虫5，23，26，迄今没有发生过感染的方法进行了详细的视觉描述。在这里，我们提出后脑普里姆25斑马鱼心室显微注射，包括我们以前的协议的修改我们的技术。 在体外研究中我们发现真菌感染的幼虫斑马鱼模型偏离和加强需要检查主机的病原体INTERAction培养皿5简化了系统的主机，而不是在复杂的环境中。

Protocol

所有斑马鱼的护理协议和实验机构动物照顾及使用委员会（IACUC）协议A2009-11-01下进行。 1。吗啉和幼虫注射菜 实验时间：*（10-15分钟） 难度：* 对于鸡蛋打针，准备在无菌水和微波的2％的琼脂糖溶液。当溶液冷却，倒入了一些额外的深培养皿（Fisher Scientific则），直到它是半满。酷冰，确保板是水?…

Discussion

这里的斑马鱼显微注射的方法不同于，Gutzman 等。在这里34我们展示通过注射到36至48 HPF幼虫的后脑心室耳泡。我们所描述的方法可以减少组织损伤后脑心室10-15酵母一致的注入。该协议产生的最初24 HPI（ 图1）造成重大杀伤力/ 5发病，整个身体蔓延的局部感染。后脑脑室不完全封闭，直到48 HPF 27，29。在此发展的巨噬细胞和中性粒细胞的早期迁移到感染?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

笔者想感谢显微注射培训，克拉丽莎亨利的意见，对加快胚胎发育和设备的使用博士卡罗尔金的实验室，和，弥敦道劳森贡献FLI1：EGFP鱼。我们感谢手稿的批判性阅读的惠勒实验室和肖恩墙成员。我们也想感谢马克Nilan鱼的保健和咨询，瑞安Phennicie和克里斯汀的Gabor在这个项目上的技术咨询。资助这项工作是由MAFES哈奇授予1 MAFES E08913-08，和国立卫生研究院的NCRR奖P20RR016463河惠勒光兄弟，研究助理。

Materials

Name of the reagent Company Catalog number Comments (optional)
Spawning tanks Aquatic habitats  2L  
1.7 mL tubes Axygen MCT-175-C
Instant Ocean Fisher Scientific S17957C  
Extra deep Petri dishes Fisher Scientific 08-757-11Z  
Standard Petri dishes VWR Scientific 25384-302
Transfer pipettes Fisher Scientific 13-711-7M  
Yeast Extract VWR Scientific 90000-726  
Peptone VWR Scientific 90000-264  
Dextrose Fisher Scientific D16-1  
Agar VWR Scientific 90000-760  
Disposable Hemocytometer VWR Scientific 82030-468  
Phosphate Buffered Saline VWR Scientific 12001-986  
Dumont Dumoxel Tweezers VWR Scientific 100501-806  
Wooden Dowels VWR Scientific 10805-018
KimWipes VWR Scientific 300053-964
Low Melt Agarose VWR Scientific 12001-722  
Agarose for injection dishes VWR Scientific 12002-102
Flaming Brown Micropipette Puller Sutter Instruments P-97
Hollow glass rods Sutter Instruments BF120-69-10 For glass rods smooth glass by heating over bunsen burner 
Pipette Storage Box Sutter Instruments BX10
MPPI-3 Injection system Applied Scientific Instrumentation MPPI-3
Back Pressure Unit Applied Scientific Instrumentation BPU  
Micropipette Holder kit Applied Scientific Instrumentation MPIP  
Foot Switch Applied Scientific Instrumentation FSW  
Micromanipulator Applied Scientific Instrumentation MM33  
Magnetic Base Applied Scientific Instrumentation Magnetic Base  
Tricaine methane sulfonate Western Chemical Inc. MS-222  
Dissecting Scope Olympus SZ61 top SZX-ILLB2-100 base  
Confocal Microscope Olympus IX-81 with FV-1000 laser scanning confocal system  
TC-7 Tissue Culture Roller drum with 14 inch test tube wheel New Brunswick Scientific  TC-7  
Imaging Dishes MatTek Corporation P24G-1.0-10-F  
Pipette tips for loading needles Eppendorf 930001007  
Plate pouring grids Adaptive Science Tools TU-1
Heated Stage Bioptechs Inc. Delta T-5
Flat Spatula VWR Scientific 82027-486
Plastic Sieves Wares of Knutsford Online 12 cm
Parafilm VWR Scientific 52858-000
Vortex Genie VWR Scientific 14216-184
16 x 150 mm Culture tubes VWR Scientific 60825-435
Nanodrop Thermo Scientific ND 2000
Phenol Red VWR Scientific 97062-478
HCl VWR Scientific 87003-216
NaCl VWR Scientific BDH4534-500GP
KCl VWR Scientific BDH4532-500GP
MgSO4 VWR Scientific BDH0246-500GP
Ca(NO3)2 VWR Scientific BDH0226-500GP
HEPES VWR Scientific BDH4520-500GP
Morpholinos GeneTools, LLC
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Tags

Non-invasive ImagingDisseminated CandidiasisZebrafish LarvaeCandida AlbicansInnate ImmunityGenetic DefectsPhagocyte NADPH OxidaseDynamics Of C. Albicans InteractionMacrophagesNeutrophilsIn Vitro StudiesIn Vivo EnvironmentCytokine LevelsExtracellular Matrix AttachmentsIntercellular ContactsModel OrganismLive Intact HostZebrafish LarvaInnate Immune Defenses
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Brothers, K. M., Wheeler, R. T. Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae. J. Vis. Exp. (65), e4051, doi:10.3791/4051 (2012).
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