Name: 啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析 (视频) | JoVE Journal
Uploaded: 2019-05-30T12:30:00.000+00:00
Duration: 8 min 23 s
Description: 学习啮齿动物疟原虫的标准化表型分析方法，涵盖野生型和转基因虫株的无性期、性血期及蚊子传播阶段。该技术为疟疾病理机制研究提供可重复的实验框架。

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5 minutes

Ahmed Aly 获得了土耳其发展部资助贝兹米亚勒姆瓦基夫大学的项目（项目编号：2015BSV036）、杜兰大学公共卫生与热带医学学院提供的资金，以及美国国立卫生研究院过敏与传染病研究所（NIH-NIAID）R21 项目（项目编号：1R21AI111058-01A1）的资助。

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<li>Aly, A. S., Lindner, S. E., MacKellar, D. C., Peng, X., Kappe, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SAP1+is+a+critical+post-transcriptional+regulator+of+infectivity+in+malaria+parasite+sporozoite+stages%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">SAP1 is a critical post-transcriptional regulator of infectivity in malaria parasite sporozoite stages.</a> Molecular Microbiology. 79, 929-939 (2011).</li>
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<li>Janse, C. J., Ramesar, J., Waters, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-efficiency+transfection+and+drug+selection+of+genetically+transformed+blood+stages+of+the+rodent+malaria+parasite+Plasmodium+berghei%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.</a> Nature Protocols. 1, 346-356 (2006).</li>
<li>Lin, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+%27gene+insertion%2Fmarker+out%27+%28GIMO%29+method+for+transgene+expression+and+gene+complementation+in+rodent+malaria+parasites%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites.</a> PLoS One. 6, e29289(2011).</li>
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</ol>

作者无任何利益冲突需要披露。

尽管小鼠疟疾模型的生活周期总体生物学特性与人类疟疾寄生虫相似，但其与人类Plasmodium物种之间仍存在诸多差异，这些差异限制了其作为可靠的in vivo模型的应用。例如，除减毒活疫苗外，所有基于亚单位疫苗、DNA疫苗及其他类型疫苗的研究在小鼠模型中均取得了良好效果，但在疟疾流行地区的人群中，这些疫苗的效果却远未达到令人满意的程度。
另一个问题是，不同小鼠品系之间，甚至同一小鼠品系来自不同供应商时，其生活周期阶段的感染性存在差异。此外，目前广泛用作首选的两种主要啮齿类疟原虫物种 体内 疟疾模型， P. berghei 和 P. yoelii，不表现出同步的红细胞期周期，这与任何人疟原虫完全不同。然而，使用小鼠疟原虫作为研究模型具有以下优势： 体内 模型的优势远远超过这些差异，而这些差异也可通过更深入地分析这些局限性的分子驱动因素加以克服。然而，这些局限性主要体现在啮齿类疟原虫的红内期寄生虫中，而在疟原虫的其他生活周期阶段中则不甚明显。
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疟原虫在全球范围内导致数亿人感染疟疾，每年造成超过60万人死亡1。人类感染由五种疟原虫引起，即 P. falciparum，P. vivax，P. ovale，P. malariae，和 P. knowlesi. 大多数临床疟疾病例死亡由 P. falciparum 在撒哈拉以南非洲1.&#160;另一种在撒哈拉以南非洲以外地区引起广泛全球发病率的人类疟原虫物种是 P. vivax2.&#160;其余三个物种的地理分布均较为局限，通常引起良性疟疾感染，但致命的除外 P. knowlesi3相关且实用的非人感染体内模型的缺乏一直是也是疟疾疫苗和药物研发的障碍。早期的疟疾药物靶向和代谢研究在很大程度上依赖于禽类疟疾模型，例如 P. gallinaceum 和 P. lophurae分别感染鸡和鸭4此后，啮齿类疟原虫种类逐渐被引入多种疫苗和药物靶向研究中，作为体内模型使用。多年来，关于啮齿类疟原虫模型在生活史各阶段的生物学特性及宿主-寄生虫相互作用与人类疟原虫相似的证据不断积累。
特别是，啮齿类动物疟疾模型对于探索和表征蚊媒阶段及红细胞前阶段的生物学特性极为重要5然而，有四种啮齿类疟原虫物种（P. berghei, P. yoelii, P. chabaudi，以及 P. vinckei）具有不同的生物学特征，其中最显著的差异体现在血液阶段6啮齿类疟原虫物种在血液期的同步性上存在差异，其中血液期阶段的 P. chabaudi 和 P. vinckei 虫株基本同步，而血液期阶段的 P. berghei 和 P. yoelii 不是6,7另一个显著差异是某些菌株中出现的血液期自我清除现象（例如, P. yoelii 17X-NL P. berghei NK65，以及 P. vinckei lentum），而同一物种的其他菌株引起的血流感染若不治疗可能会致命（P. yoelii 17X-L, P. berghei ANKA，以及 P. chabaudi AS）。此外， P. yoelii 17X-NL 菌株和 P. berghei ANKA 菌株优先侵染网织红细胞8,9,10,11，尽管这些特征 P. yoelii 和 P. berghei 菌株并非严格的生长需求12,13,14因此，在感染这些寄生虫的血液阶段之前，需先用苯肼处理小鼠，以提高寄血症和配子体血症水平，从而满足蚊虫感染所需的条件 P. berghei ANKA 小鼠品系及用于 P. yoelii 17X-NL15,16,17,18,19.
不同啮齿类疟原虫物种之间的蚊期发育也存在差异，其中最显著的是蚊期发育所需的最适温度和时间，以及子孢子的长度5,6,20。在啮齿类疟原虫的红细胞前体阶段，差异包括对感染性子孢子接种最敏感的啮齿类动物种类和品系、在敏感品系中实现接种所需的子孢子数量、体外肝期发育实验所需的哺乳动物细胞类型，以及完成肝期发育所需的时间5,21,22,23,24,25,26,27,28,29,30。
尽管存在这些变异，啮齿类动物疟原虫早期仍是应用反向遗传学方法的首选模型，因为其耗时较少、资源需求较低，且成功率较高31。事实上，在多年间，啮齿类动物疟原虫模型是最理想的模型，且在许多情况下是唯一可用于功能性表征蚊子和肝脏阶段表达基因的模型。
鉴于反向遗传学方法在啮齿类疟疾模型中的广泛应用和可操作性，已有多种不同方法被用于分析转基因寄生虫生活史阶段的表型，尤其是血液阶段。然而，其中一些方法存在不一致之处；例如，比较腹腔注射（IP注射）后血液阶段寄生虫的感染情况时，注射的寄生虫可能被引流至腹膜淋巴结，并由此进入血液循环，因此注入的寄生虫并非均等地分布于血液中；又如，比较经过不同次数连续血液传代或G代数（G number）的克隆在蚊媒传播中的差异（这可能影响配子体形成32,33）；再如，将转基因寄生虫直接与从未经历电穿孔和药物阳性筛选的 naïve 野生型（WT）寄生虫进行比较，以及对雄性配子体出丝现象（exflagellation）进行的各种非标准化评估。因此，建立简单易行且标准化的操作流程，以适应啮齿类疟原虫物种在生物学上的变异性，对任何类型转基因或野生型啮齿类疟原虫在血液及蚊体内的表型分析至关重要。
本文报告了一种标准化的详细实验方案，用于转基因或野生型P. yoelii和P. berghei疟原虫血液阶段及蚊媒生活周期阶段的表型分析。 该方案同样适用于P. chabaudi和P. vinckei疟原虫。

<table><tbody><tr><td>肝素</td><td>Sigma</td><td>375095-100KU</td><td></td></tr><tr><td>黄尿酸</td><td>Sigma</td><td>D120804-5G</td><td></td></tr><tr><td>次黄嘌呤</td><td>Sigma</td><td>H9377-25G</td><td></td></tr><tr><td>阿尔塞弗溶液</td><td>Sigma</td><td>A3551-500ML</td><td></td></tr><tr><td>碳酸氢钠</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>苯肼</td><td>Sigma</td><td>P26252-5G</td><td></td></tr><tr><td>甘油</td><td>Sigma</td><td>G5516-500ML</td><td></td></tr><tr><td>吉姆萨染液</td><td>Sigma</td><td>GS1L-1L</td><td></td></tr><tr><td>26 G x 3/8 精密滑动针&amp;nbsp;</td><td>Becton Dickinson</td><td>305110</td><td></td></tr><tr><td>1 mL 结核菌素注射器，26 G x 3/8</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>1 cc 胰岛素注射器，U-100 27 G</td><td>Becton Dickinson</td><td>329412</td><td></td></tr><tr><td>异氟烷，USB</td><td>Piramal</td><td>2667- 46- 7</td><td></td></tr><tr><td>PBS，pH 7.4</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>RPMI</td><td>Gibco</td><td>22400105</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995065</td><td></td></tr><tr><td>青霉素/链霉素</td><td>Gibco</td><td>10378016</td><td></td></tr><tr><td>胎牛血清</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>玻璃纤维棉</td><td>Corning</td><td>3950</td><td></td></tr></tbody></table>

啮齿类疟原虫无性与有性血液阶段及蚊期阶段的表型分析

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

近年来，遗传学和系统生物学技术的进步加深了我们在分子水平上对疟原虫生物学的理解。然而，可用于疫苗和化学治疗开发的有效疟原虫靶点仍然有限。这主要归因于缺乏相关且实用的 体内 人类感染模型 疟原虫 物种，尤其是对于 P.&#160;恶性疟原虫 和 P. vivax因此，啮齿类疟原虫种类被广泛用作疟疾疫苗、药物靶向、免疫反应以及保守基因功能表征研究中实用的替代体内模型 疟原虫属 基因。事实上，啮齿类动物疟疾模型已被证明具有不可估量的价值，尤其在研究蚊媒传播和肝期生物学方面，对免疫学研究也至关重要。然而，在评估转基因与野生型无性期及有性期血液阶段寄生虫表型的方法上仍存在差异。&#160;这些差异的实例包括静脉注射的选择 与 啮齿类动物腹腔感染红内期寄生虫及雄性配子出丝现象的评估。本文详细阐述了用于评估表达报告基因或野生型啮齿类疟原虫物种的无性与有性红内期表型的标准化实验方法。同时，我们也详细描述了在蚊体内评估疟原虫各蚊期阶段（配子、动合子、卵囊和子孢子）表型的方法。 按蚊属 蚊媒。本文针对致死性和非致死性菌株，对这些方法进行了详细说明和简化。 P. berghei 和 P. yoelii 但经过一些调整后也可应用于 P. chabaudi 和 P. vinckei 啮齿类疟原虫物种

将反向遗传学工具和技术成功应用于疟原虫，彻底改变了疟疾研究领域，使其能够添加、删除或修饰多种疟原虫的特定基因组片段 疟原虫 物种39重要的是，已鉴定出可缺失的基因组位点，并通过双同源重组技术成功用于在啮齿类和人类疟原虫中引入荧光蛋白标记，以确保在所有生活周期阶段中的稳定表达40,41,42一种此类野生型样转基因寄生虫的实例是 Py230p(-) 寄生虫，由本实验室构建，其血液阶段和蚊媒生命周期阶段的发育均未表现出明显缺陷15,16,17这些转基因报告子寄生虫在强且组成型启动子的调控下表达 eGFP， PyHSP7...

由于啮齿类疟原虫在生命周期和生物学特性上与人类疟原虫具有显著相似性，啮齿类疟疾模型已成为疟疾研究不可或缺的工具。本文中，我们对野生型和转基因啮齿类疟原虫进行表型分析时所使用的一些最重要技术进行了标准化。

遗传学和系统生物学技术的最新进展推动了我们在分子水平上对疟原虫生物学的理解。

由于啮齿类疟原虫在生命周期和生物学特性方面与人类疟原虫具有显著相似性，啮齿类疟原虫模型已成为疟疾研究中不可或缺的工具。这些标准方法有助于回答疟疾研究领域中的关键问题，例如如何对野生型和转基因啮齿类疟原虫进行表型分析。本实验操作将由我实验室的研究生Gozde Deveci和Ilknur Yilmaz进行示范。首先快速解冻冷冻保存的寄生虫储备液，并立即使用配备26号针头的1毫升注射器，将约200微升储备液经腹腔注射至每种基因型至少两只供体小鼠体内。注射24小时后，用24或26号针头刺破第一只小鼠的尾部，将血滴收集到载玻片上。用另一张载玻片的短边迅速将血液在第一张载玻片上推片。 当血液完全干燥后，将玻片浸入100%甲醇中固定至少一分钟。将固定后的样本用吉姆萨染液染色10至15分钟，随后用单蒸水或双蒸水洗涤一次或两次。待玻片干燥后，在光学显微镜下使用100倍油镜观察，计数每个网格中感染的红细胞总数。感染后两到三天，将寄生血症水平达到0.1%至1%的小鼠麻醉并固定于仰卧位，用镊子提起胸骨基部附近的皮肤，以便进行皮肤切口。在切口处将皮肤拉开，暴露膈肌和腹腔上部，用镊子夹住肋骨基部，同时剪开膈肌以进入胸腔。将肋骨向后固定，暴露心脏，使用一支含有至少20 µL肝素并装有26号针头的1 mL注射器，轻轻穿刺心脏组织。缓慢回抽针栓，收集约一毫升血液，并将血液注入微量离心管中。待所有血液采集完毕后，在红色加热灯下预热受体小鼠，并用含有适当剂量寄生虫的胰岛素注射器（每只受体小鼠配备一支装有27号针头的胰岛素注射器）进行准备。随后将第一只受体小鼠放入固定器中，将一剂感染的红细胞注入其扩张的尾静脉。进行蚊虫饲血时，选取饥饿8至12小时、羽化后4至7日龄的雌性斯氏按蚊（Anopheles stephensi）或冈比亚按蚊（A. gambiae），让其在感染程度最高、配子体出丝率最强的麻醉小鼠身上饲血，饲血时间不少于15分钟，以确保充分吸血。为测定每只蚊虫中卵囊子孢子的数量，将20至30只蚊虫置于冷冻环境中安乐死，冷冻时间不少于10分钟；随后使用双目光学解剖显微镜及两枚26或27号细针，在载玻片上的适当解剖液中解剖蚊虫中肠。解剖中肠时，先用一根针固定蚊虫腹部下端，再用另一根针轻轻向上推动胸腹部交界处，力度需轻微，直至胸部与腹部分离，并从中垂下一条白色中肠组织。使用之前用于上推胸部的同一根针，从食道端开始剪断中肠，从而分离并展开中肠。用针将解剖好的中肠从解剖液中提起，转移至含有 200 微升 RPMI 的离心管中。当所有中肠均解剖并收集完毕后，将装有中肠的离心管放入离心机，在 700 × g 条件下离心 1 分钟。然后用研杵将收获的组织研磨两次。取12 µL稀释后的组织溶液转移至血细胞计数板，于室温孵育5分钟，使内容物沉降。随后在相差显微镜下以40×放大倍数观察，确定卵囊子孢子的平均数量。为

phenotypic-analysis-rodent-malaria-parasite-asexual-sexual-blood

Research

科研

JoVE Journal

Immunology and Infection

免疫学与感染

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传播疟疾的蚊子具有一些重要的流行病学特征，这些特征只能通过分子技术进行检测。我们利用基于 MALDI-TOF 的 SNP...

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我们描述了一种实验性脑型疟疾的小鼠模型，并展示了如何追踪炎症和微血管病理变化 体内 使用磁共振成像。...

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对疟原虫蚊期阶段进行详细研究，对于设计有效的阻断传播策略至关重要。本方案展示了如何有效培养具有感染性的配子体，并将这些配子体喂食给蚊子，以获得恶性疟原虫（P.

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Ookluc：一种用于鉴定阻断传播化合物的Plasmodium berghei菌株

学习啮齿动物疟原虫的标准化表型分析方法，涵盖野生型和转基因虫株的无性期、性血期及蚊子传播阶段。该技术为疟疾病理机制研究提供可重复的实验框架。

啮齿类疟原虫无性与有性血液阶段及蚊期阶段的表型分析

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