Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.
Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.
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DOI：
10.1371/journal.pone.0007881
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发表时间：
2009-11-18
期刊：
影响因子：
3.7
通讯作者：
Franke-Fayard BM
中科院分区：
文献类型：
--
作者：
Ploemen IH;Prudêncio M;Douradinha BG;Ramesar J;Fonager J;van Gemert GJ;Luty AJ;Hermsen CC;Sauerwein RW;Baptista FG;Mota MM;Waters AP;Que I;Lowik CW;Khan SM;Janse CJ;Franke-Fayard BM
寄生虫感染率低以及监测细胞内发育所需的复杂方法阻碍了对培养细胞中实验动物肝脏中疟原虫发育的定量分析。因此，与血液阶段相比，寄生虫生命周期的这一重要阶段的研究很少，例如在筛选抗疟疾药物方面。在这里，我们报告了使用转基因伯氏疟原虫寄生虫 PbGFP-Luccon，表达生物发光报告蛋白荧光素酶，使用实时发光成像来可视化和量化培养物和活体小鼠肝细胞中的寄生虫发育。通过实时成像或使用酶标仪对培养的肝细胞进行基于报告寄生虫的定量，与已建立的定量 RT-PCR 方法有很好的相关性。首次在活体小鼠的全身中观察到疟原虫的肝脏阶段，我们能够使用 2D 成像区分小鼠每个肝脏中少至 1-5 个感染的肝细胞，并通过 3D 成像识别单个感染的肝细胞。全身成像对肝脏感染的分析与提取肝脏的定量 RT-PCR 分析具有良好的相关性。基于发光的分析各种药物对体外肝细胞感染的影响表明，该方法可有效用于体外筛选针对疟原虫肝阶段的化合物。此外，通过分析伯氨喹和他非诺喹的体内作用，我们证明了实时成像在评估肝脏中寄生虫药物敏感性方面的适用性。与PCR方法相比，通过实时成像对肝脏阶段发育进行定量分析的简单性和速度，以及无需手术即可分析活体小鼠肝脏发育的可能性，为研究疟原虫肝脏感染以及验证药物和疫苗对疟原虫肝脏阶段的影响开辟了新的可能性。
The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.
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