Université de Toulouse, UPS, LPSNPR (Laboratoire pharmacochimie des substances naturelles et pharmacophores redox), Toulouse, France.
Biosens Bioelectron. 2010 Aug 15;25(12):2566-72. doi: 10.1016/j.bios.2010.04.021. Epub 2010 Apr 21.
The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedance spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulated macrophages. This production was not observed in the case of Leishmania-infected macrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC)(2) as NO radical spin trap were carried out.
生理变化由外部刺激引起，可以作为研究细胞病原体感染和药物作用的参数。在分析方法中，阻抗分析通过研究形态变化、生理状态的改变、带电或氧化还原物质的产生，而不干扰体外细胞代谢和标记，有可能深入了解细胞行为。本工作描述了使用电化学阻抗谱通过建模阻抗谱图（奈奎斯特图）在适当的等效电路中简单地监测，在适当的等效电路中简单地监测，响应寄生虫感染利什曼原虫或脂多糖（LPS）处理的鼠巨噬细胞系（RAW 264.7）的变化。这些结果表明电化学阻抗谱能够区分两种相反的细胞反应，这两种反应与两种不同的刺激有关，一种是由寄生虫的内化引起的，另一种是由细菌成分的激活引起的。事实上，这项研究从电的角度表征了由 LPS 刺激的巨噬细胞中诱导型一氧化氮合酶内源性和大量产生的细胞外 NO 自由基。在利什曼原虫感染的巨噬细胞中没有观察到这种产生，为了生存和繁殖，寄生虫本身具有可能干扰 NO 产生的机制。在这种情况下，只观察到 ROS 的细胞内产生。为了证实这些解释，使用 ROS（活性氧）荧光探针 2'，7'-二氯二氢荧光素二乙酸盐进行共聚焦显微镜分析，并使用 Fe(DETC)(2)作为 NO 自由基自旋捕获物进行电子顺磁共振实验。
