Name: zebrafish animal model for the study of allergic reactions in response to tick saliva biomolecules
Uploaded: 2022-09-16T14:10:00
Description: Watch this Scientific Journal Video about Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules at JoVE.com

我们要感谢SaBio集团的成员在实验设计和与鱼类实验设施的技术援助方面的合作，以及Juan Galcerán Sáez（IN-CSIC-UMH，西班牙）提供斑马鱼。这项工作得到了西班牙科学和创新部/MCIN/AEI/10.13039/501100011033和EU-FEDER（Grant BIOGAL PID2020-116761GB-I00）的支持。Marinela Contreras由西班牙创新与大学科学部资助，授予IJC2020-042710-I。

此处描述的所有方法均已获得卡斯蒂利亚拉曼恰大学动物实验伦理委员会的批准，该研究“评估对灭活牛支原体疫苗的免疫反应和斑马鱼型号PR-2017-05-12中的海洋支原体挑战”。
从实验室菌落获得蜱虫，通过PCR检测菌落中蜱虫的代表性样本，以确认不存在病原体，并在捷克共和国捷克科学院生物中心寄生虫学研究所（IP BC CAS）进行所有动物实验均按照捷克共和国?...

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斑马鱼是一种具有成本效益且易于处理的模型，也是研究免疫反应的分子机制，病原体疾病，新型药物测试以及疫苗接种和预防感染的非常可行的工具33，34，35。对斑马鱼行为的研究很有用，因为以前的研究发现，一些鱼类在受到压力时在水箱底部保持不动，这会影响它们的食物消耗，吃得更少;此外，它们移动时的曲折也可?...

蜱虫是引起疾病的病原体的载体，也是过敏反应的原因，影响全球人类和动物的健康1，2。在蜱虫摄食期间，蜱唾液中的生物分子，尤其是蛋白质和脂质，有助于这些体外寄生虫的摄食，避免宿主防御3。一些具有聚糖Galα1-3Galβ1-（3）4GlcNAc-R（α-Gal）修饰的唾液生物分子导致蜱叮咬后产生高抗α-Gal IgE抗体水平，仅在某些个体中，这被称为α-Gal综合征（AGS）4。这是一种与 IgE 介导的过敏相关的疾病，可能导致对蜱叮咬、非灵长类哺乳动物肉类消费和某些药物（如西妥昔单抗5）的过敏反应。对α-Gal的反应通常是严重的，有时可能是致命的6，7，8，9，10，11，12，13，14，15。
α-Gal存在于所有哺乳动物中，除了旧世界的猴子，猿类和人类，它们没有合成α-Gal13的能力。然而，细菌和原生动物等病原体在其表面表达这种聚糖，这可以诱导产生大量的抗α-Gal IgM / IgG抗体，并且可能是针对这些病原体的保护机制16，17。然而，抗α-Gal抗体的产生增加了发生IgE介导的抗α-Gal过敏的风险7，13。人类产生的天然抗α-Gal抗体，主要是IgM / IgG亚型，可能与肠道微生物群中存在的这种修饰有关16。AGS可能是一个具有挑战性的临床诊断，因为目前的主要诊断方法是基于迟发性过敏反应的临床病史，特别是与食物过敏（即瘙痒，局部荨麻疹或复发性血管性水肿到全身过敏反应，荨麻疹和胃肠道症状）和IgE抗α-Gal抗体水平的测量9相关。目前的研究结果表明，蜱叮咬是AGS18，19出现的主要风险之一，蜱虫叮咬后IgE水平增加20倍或更多至α-Gal19，AGS20，21，22患者的蜱叮咬史，AGS患者中存在对蜱抗原反应的抗体19， 抗α-Gal IgE与抗蜱IgE水平19，23密切相关，但需要进一步的研究来评估哪些生物分子实际参与其中。
此外，另一种可能的情况是患者对蜱叮咬和高水平的抗α-Gal IgE抗体表现出强烈的过敏反应，但对哺乳动物肉类消费有耐受性12。因此，哺乳动物肉类过敏可能是一种特殊类型的蜱叮咬相关过敏。与AGS相关的主要蜱种包括美洲蛱（美国），雕塑蜱（巴西），睾丸蜱和长角血蜱（日本），硬蜱（澳大利亚）和硬蜱（欧洲莱姆疏螺旋体病的主要媒介）11，24。 
唯一用于评估与蜱叮咬相关的IgE产生的模型是用基因修饰的小鼠模型α-1，3-半乳糖基转移酶敲除（α-Gal KO）小鼠25，26，因为与其他哺乳动物一样，小鼠也在蛋白质和脂质上表达α-Gal，并且不会产生IgE到α-Gal。然而，斑马鱼（Danio rerio）是应用于哺乳动物的生物医学研究的有用模型，因为它与哺乳动物有许多解剖学上的相似之处，并且像人类一样，也无法合成α-Gal。由于α-Gal不是在斑马鱼中自然产生的，因此它是一种经济实惠的模型，易于操作，并且允许高样本量用于研究α-Gal相关的过敏反应。
在这项研究中，斑马鱼被用作模式生物来表征和描述局部过敏反应、行为模式以及与经皮对蜱唾液26，27 和随后的哺乳动物肉类消费的反应相关的分子机制。为此，通过皮内注射将鱼暴露于蜱唾液中，然后喂食狗饲料，其中含有适合动物使用的哺乳动物肉类衍生产品，其中含有α-Gal27，然后评估可能的相关过敏反应。该方法可用于研究与过敏过程相关的其他生物分子，特别是与AGS相关的生物分子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL tube</td>
			<td>VWR</td>
			<td>525-0990</td>
			<td></td>
		</tr>
		<tr>
			<td>All Prep DNA/RNA</td>
			<td>Qiagen</td>
			<td>80284</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquatics facilities</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Disection set</td>
			<td>VWR</td>
			<td>631-1279</td>
			<td></td>
		</tr>
		<tr>
			<td>Dog Food - Red Classic</td>
			<td>Acana</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plates-96 well</td>
			<td>Thermo Fisher Scientific</td>
			<td>10547781</td>
			<td></td>
		</tr>
		<tr>
			<td>Gala1-3Gal-BSA 3 (&#945;-Gal)&#160;</td>
			<td>Dextra</td>
			<td>NGP0203</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript Reverse Transcription Supermix</td>
			<td>Supermix</td>
			<td>1708840</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter syringes</td>
			<td>Hamilton</td>
			<td>7638-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-50TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>pilocarpine hydrochloride&#160;</td>
			<td>Sigma</td>
			<td>P6503</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip P10&#160;</td>
			<td>VWR</td>
			<td>613-0364</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip P1000</td>
			<td>VWR</td>
			<td>613-0359</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium food tropical fish</td>
			<td>DAPC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge Animal Holder&#160;</td>
			<td></td>
			<td></td>
			<td>Made from scrap foam</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler Real-Time PCR</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methanesulphonate (MS-222)</td>
			<td>Sigma</td>
			<td>E10521</td>
			<td></td>
		</tr>
	</tbody>
</table>

zebrafish animal model for the study of allergic reactions in response to tick saliva biomolecules

蜱是通过病原体传播引起疾病的节肢动物媒介，其叮咬可能与影响全球人类健康的过敏反应有关。在一些个体中，蜱叮咬诱导了针对聚糖Galα1-3Galβ1-（3）4GlcNAc-R（α-Gal）的高水平免疫球蛋白E抗体。由蜱唾液中存在的含有聚糖α-Gal的糖蛋白和糖脂介导的过敏反应与α-Gal综合征（AGS）或哺乳动物肉类过敏有关。斑马鱼（Danio rerio）已成为研究不同病理学的广泛使用的脊椎动物模型。在这项研究中，斑马鱼被用作研究α-Gal和哺乳动物肉类消费反应的模型，因为像人类一样，它们不合成这种聚糖。为此，评估了对 硬蜱 蜱唾液和哺乳动物肉类消费的反应行为模式和出血性过敏型过敏反应。这种实验方法允许获得支持斑马鱼动物模型的有效数据，用于研究包括AGS在内的蜱传过敏。

这里介绍的方案基于先前发表的实验27，30的几个方面以及在我们的实验室进行的结果，其中斑马鱼模型被建立并验证用于研究AGS和对α-Gal的免疫反应，因为人类和斑马鱼都不会合成这种分子13。该模型允许表征和评估各种过敏反应，这是宿主对蜱唾液的反应（图4）及其在AGS中的影响的结果。此外，在鱼中观察到?...

Watch this Scientific Journal Video about Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules at JoVE.com

Zebrafish Animal Model for the Study of Allergic Reactions in Response to Tick Saliva Biomolecules

在这里，斑马鱼（Danio rerio）被用作模型，通过评估对蜱唾液和哺乳动物肉类消费的过敏反应来研究与α-Gal综合征（AGS）相关的过敏反应和免疫反应。

斑马鱼动物模型，用于研究对蜱唾液生物分子的过敏反应

Ticks are arthropod vectors that cause disease by pathogen transmission and whose bites could be related to allergic reactions impacting human health ...

问题是为什么只有一些暴露于蜱叮咬的人会出现过敏反应。对我们来说，阐明蜱唾液中的生物分子以及涉及这一过程的免疫机制是解决这个问题的关键。α-Gal综合征的斑马鱼动物模型再现了人类对蜱唾液的反应的行为模式和过敏反应。这种实验方法促进了对蜱 - 宿主分子相互作用的理解，其中我们与α-Gal综合征相关的冲突以及与α-Gal抗体反应相关的合作，这些反应可以防止病原体感染。为了用蜱虫唾液处理鱼，将成年斑马鱼分为三个性别平衡的组。使用商业Gala1-3Gal-BSA 3或α-Gal作为阳性对照，PBS作为阴性对照。对于唾液提取，使用半充血、无病原体的雌性蜱虫喂食豚鼠六到七天。用50微升注射器和0.33毫米针头在PBS中的2%盐酸毛果芸香碱溶液以pH 7.4处理蜱虫。使用安装在微量移液器上的 10 微升吸头从蜱下口收集唾液。将唾液放入冰上的1.5毫升管中，并将其储存在零下80摄氏度。按照制造商的建议，使用BCA蛋白质检测试剂盒确定唾液蛋白浓度以确定要注射到鱼中的蛋白质量。将斑马鱼饲养在 27 摄氏度的流通水系统中，明暗循环为 14 至 10 小时。接下来，每组选择10条鱼，雌雄比例相似，体重相似注射。通过浸入0.02%甲烷磺酸三卡因中短暂麻醉鱼。用镊子或手小心地将麻醉的鱼放在湿海绵上的半侧，尾鳍在右侧以相同的方向注射化合物以控制病变。用装有1厘米，29号针头的100微升注射器在肌肉中皮内注射鱼群，距离尾鳍5毫米，与鱼体成45度角。注射后，将处理过的鱼放回淡水箱中，无需麻醉即可恢复。每天上午 9：30 喂鱼两次。下午 1：30，每条鱼喂 50 至 70 微克干鱼，直到第二天。然后用研钵和研杵捣碎狗粮，从处理注射后的第二天到第八天的实验结束喂鱼。对于样本收集，将安乐死的鱼固定在带有销钉的石蜡板上。当鳃仍然用血液冲洗时，使用装有一厘米，29号针头的0.5毫升注射器从鱼的鳃血管中收集血清。将收集的血清储存在 1.5 摄氏度的 20 毫升管中直至使用。用手术刀刀片将鱼切成矢状，并评估内部病变。然后将每条鱼的肠和肾收集在单独的 1.5 毫升管中。使用 RNA 纯化试剂盒从肠道和肾脏中提取总 RNA。根据制造商的说明，分析与免疫反应相关的基因的表达，使用RT-qPCR的逆转录混合物进行定量逆转录聚合酶链反应或RT-qPCR。对斑马鱼 GAPDH 的 mRNA cT 值进行归一化，并使用具有不等方差的学生 t 检验在组间进行比较。通过 ELISA 测定可识别血清样品中 α-Gal 的 IgM 抗体滴度。使用酶标仪将抗体滴度记录为光密度450纳米值，并使用方差不等的学生t检验在组间进行比较。该斑马鱼模型允许表征和评估由于宿主对蜱唾液的反应及其对α-Gal综合征或AGS的影响而导致的各种过敏反应。此外，与对照鱼相比，在蜱唾液处理的鱼中观察到诸如缓慢游泳，躺在水箱底部以及不进食，振动或曲折运动等行为的改变。在用蜱唾液处理的鱼中观察到过敏反应的显着发生率，显示出快速脱敏和耐受性。用蜱唾液处理的鱼的行为变化比仅用α-Gal处理的鱼更明显。通过RT-PCR对代表性免疫标志物的表达进行额外分析。结果显示，与对照组相比，用唾液和α-Gal处理的鱼的斑马鱼组之间存在差异。此外，用唾液和α-Gal处理的斑马鱼产生了针对α-Gal的IgM抗体，其水平高于用PBS处理的鱼。注射治疗，评估过敏反应和行为，以及报告过敏反应造成的死鱼数量是该方案最重要的方面。使用mRNA，蛋白质和微生物组水平的组学技术来表征对蜱唾液的反应，使我们能够识别该过程所涉及的关键生物分子。

Research

JoVE Journal

Immunology and Infection

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

在仓鼠的背部皮肤：皮肤利什曼病Antileishmanial药物筛选的一个有用的模型

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

呼吸爆发的响应为先天免疫健康的斑马鱼中的指标量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

利用RNA干扰调查的先天免疫反应的小鼠巨噬细胞

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...