啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析
Summary
由于啮齿动物疟原虫的生命周期和生物学与人类疟原虫有着惊人的相似性,啮齿动物疟疾模型已成为疟疾研究不可或缺的一部分。在这里,我们标准化了野生型和转基因啮齿动物疟疾物种的类名分析中使用的一些最重要的技术。
Abstract
遗传学和系统生物学技术的最新进展促进了我们对疟原虫生物学在分子水平上的理解。然而,疫苗和化疗开发的有效疟原虫目标仍然有限。这主要是由于人类疟原虫物种缺乏相关和实用的体内感染模型,最显著的是恶性疟原虫和P.vivax。 因此,啮齿动物疟疾物种在体内被广泛用作疟疾疫苗、药物靶向、免疫反应和保存的疟原虫基因的功能表征研究的实用替代物。事实上,啮齿动物疟疾模型已被证明是无价的,特别是对于探索蚊子传播和肝阶段生物学,并且对于免疫学研究是必不可少的。然而,用于评估转基因和野生类无性及性血期寄生虫的表型的方法存在差异。这些差异的例子包括选择静脉注射与内腹感染啮齿动物与血期寄生虫,以及评估雄性Gamete灭菌。本文详细介绍了标准化的实验方法,以评估转基因寄生虫中无性血和性血阶段的表型,表达报告基因或野生型啮齿动物疟原虫物种。我们还详细介绍了评估疟原虫蚊媒内疟原虫阶段(配子、卵母细胞、卵泡和孢子菌)的表型的方法。 这些方法在这里详细和简化,用于P.berghei和P.yoelii的致命和非致命菌株,但也可用于对P.chabaudi和P.vinckei啮齿动物疟疾物种进行一些调整。
Introduction
疟原虫在全世界造成数以亿计的疟疾感染,每年有60多万人死于疟疾。人类感染是由五种疟原虫引起的,即恶性疟原虫、疟原虫、疟原虫、疟原虫和疟原虫。 大多数临床疟疾死亡是由撒哈拉以南非洲地区恶性疟原虫引起的。另一种在撒哈拉沙漠以南的非洲地区引起广泛全球病症的人类疟原虫是P.vivax2。其他三个物种都更受地理限制,并引起良性疟疾感染,除了致命的P. 缺乏相关和实际的非人类体内感染模型一直是而且仍然是疟疾疫苗和药物开发的障碍。早期的疟疾药物靶向和代谢研究已经广泛依赖于禽流感模型,如胆汁和P.lophurae,分别感染鸡和鸭,分别4。此后,各种疫苗和药物靶向研究逐渐引入啮齿动物疟疾物种,作为体内模型。多年来,啮齿动物疟疾模型与人类疟疾物种的生物学和宿主-寄生虫相互作用的相似性证据不断积累。
特别是,啮齿动物疟疾模型对于探索和描述蚊子生物学和红细胞前第5阶段极为重要。然而,有四种啮齿动物疟疾物种(P.berghei,P.yoelii,P.chabaudi和P.vinckei)具有不同的生物特征,其中最显著的是血液阶段6。 疟疾的疟疾物种在血液阶段的同步性上有所不同,其中P.chabaudi和P.vinckei菌株的血期大部分是同步的,而P.berghei和P.yoelii的血期不是6,7.另一个显著区别是某些菌株(例如,P. yoelii 17X-NL、P. berghei NK65 和P. vinckei lentum)的血液阶段的自我清除,而其他菌株的血液感染如果得不到治疗,同一物种的菌株可能是致命的(P.yoelii 17X-L,P. berghei ANKA 和P. chabaudi AS)。此外,P.yoelii 17X-NL 菌株和P. berghei ANKA 菌株优先入侵视网膜 8、9、10、11,尽管 P 的这些特征。尤利和宝海菌株不是严格的生长要求12,13,14。因此,在感染这些寄生虫的血液阶段之前,小鼠使用苯乙酰乙酰胺进行治疗,以增加P.berghei ANKA菌株和P.yoelii的蚊子感染所需的寄生虫血症和肌细胞血症17X-NL15,16,17,18,19。
不同啮齿动物疟疾物种之间也存在蚊子阶段发育的差异,其中最显著的是最佳蚊子阶段发育所需的温度和时间以及孢子菌长度5、6 20.在啮齿动物疟疾菌群的红细胞前阶段,差异包括最易感染孢子菌的啮齿动物种类和菌株,易感啮齿动物菌株接种所需的孢子菌数量,体外肝阶段发育测定所需的哺乳动物细胞类型,以及完成肝阶段发育的时间5、21、22、23、24、25 ,26,27,28,29,30.
尽管存在这些变异性,啮齿动物疟原虫在早期是应用反向遗传方法的有利模型,因为它们较少耗费时间和资源,成功概率高31。事实上,啮齿动物疟疾模型是最好的模型,在许多情况下,是唯一的模型,可用于功能特征基因表达在蚊子和肝脏阶段多年。
鉴于反向遗传方法在啮齿动物疟疾模型中的流行性和可得性,已采用多种不同的方法分析转基因寄生虫生命周期阶段的表型,特别是血液阶段。然而,其中一些方法不一致;例如,比较注射IP后血期寄生虫的感染(可能排到围肠淋巴结,从那里进入血液;因此,注射的寄生虫在血液中不会同样地进入),将克隆的蚊子传播与不同数量的序列血阶段转移或G数(可能影响细胞生成32,33)进行比较,或将转基因寄生虫直接与幼稚的野生型(WT)进行比较从未接受电穿孔和阳性药物选择以及各种非标准化的雄性配药除虫评价的寄生虫。因此,对血液和蚊子中任何类型的转基因或WT啮齿动物疟原虫进行类称分析,以适应啮齿动物疟疾的生物变异性,使简单易遵循的协议标准化至关重要。寄生虫物种。
本文报告转基因或野生P.yoelii和P.berghei寄生虫的血液和蚊子生命周期阶段的标准化、详细的实验方案。这些协议也适用于P.chabaudi和P.vinckei寄生虫。
Protocol
Representative Results
Discussion
尽管小鼠疟疾模型在一般生物学中与人类疟原虫的生命周期相似,但鼠疟疾模型与人类疟原虫物种也有许多不同,这将限制其作为可靠体内模型的使用。例如,除了作为疫苗的活减毒寄生虫外,所有带有亚单位、DNA和其他疫苗的疫苗研究在小鼠模型中都取得了优异的结果,但在生活在流行地区的人类中,结果远不能令人满意。
另一个问题是生命周期阶段感染性从一个小?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Ahmed Aly 得到土耳其发展部 2015BSV036 赠款给 Bezmialem Vakif 大学的资金支持,以及杜兰大学公共卫生和热带医学学院提供的资金,以及 NIH-NIAID 为 R21Grant 提供的资金1R21AI111058-01A1。
Materials
| Heparin | Sigma | 375095-100KU | |
| Xanthurenic acid | Sigma | D120804-5G | |
| Hypoxanthine | Sigma | H9377-25G | |
| Alsever's solution | Sigma | A3551-500ML | |
| Sodium Bicarbonate | Sigma | S5761-500G | |
| Phenylhydrazine | Sigma | P26252-5G | |
| Glycerol | Sigma | G5516-500ML | |
| Giemsa | Sigma | GS1L-1L | |
| 26G x 3/8 Precision Glide Needle, | Becton Dickinson | 305110 | |
| 1 ml TB Syringe, 26G x 3/8 | Becton Dickinson | 309625 | |
| 1 cc Insulin Syringe, U-100 27G | Becton Dickinson | 329412 | |
| Isoflurane, USB | Piramal | 2667- 46- 7 | |
| PBS, pH 7.4 | Gibco | 10010049 | |
| RPMI | Gibco | 22400105 | |
| DMEM | Gibco | 11995065 | |
| Pencillin/ Streptomycin | Gibco | 10378016 | |
| Fetal Bovine Serum | Gibco | 10082147 | |
| Fiber Glass Wool | Corning | 3950 |
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