A novel flow cytometric assay for quantitating adherence of Helicobacter pylori to gastric epithelial cells
A novel flow cytometric assay for quantitating adherence of Helicobacter pylori to gastric epithelial cells
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DOI：
10.1016/s0022-1759(98)00018-0
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发表时间：
1998-04-01
影响因子：
2.2
通讯作者：
Hawkey, CJ
中科院分区：
文献类型：
--
作者：
Logan, RPH;Robins, A;Hawkey, CJ
粘附可能是幽门螺杆菌的重要毒力因素。目前可用于定量依从性的方法非常耗时并且容易出现观察者错误。一种新的细菌荧光标记直接技术已被开发出来，通过荧光激活细胞分选（FACS）来定量幽门螺杆菌与上皮细胞的粘附。 H. pylori、H. Mustelae、H. cinaedi 和 H. fennelliae 的典型菌株在肉汤培养物中微需氧生长 24 小时，并通过在 37°C 下与羧基荧光素二乙酸琥珀酰亚胺酯 (CFDA-SE) 一起孵育进行荧光标记。洗涤去除多余的 CFDA-SE 后，将细菌与胃上皮细胞在 37°C 下共孵育（比例 10:1）。摄氏度长达 24 小时。洗涤去除非粘附细菌后，用 EDTA (2 mM) 分离上皮细胞，并用甲醛固定以进行流式细胞术。根据粘附幽门螺杆菌的细胞比例和每个细胞粘附细菌的平均数量来定量粘附。所有幽门螺杆菌菌株均粘附于胃型上皮细胞。结合细菌的细胞比例为 40-99%，每个细胞的细菌数量为 1-50，两者均与显微镜相关（分别为 r = 0.6 和 r = 0.8，n = 35）。时程研究表明幽门螺杆菌在 90 分钟内结合饱和。对于鼬鼠、灰鼠和芬氏菌，结合细菌的细胞比例为 5-15%，每个细胞的平均细菌数 < 4。通过与多克隆抗血清预孵育或使用针对胃粘液潜在结合表位的寡糖，可以部分阻断幽门螺杆菌与上皮细胞的结合。使用 CFDA-SE 荧光标记幽门螺杆菌并结合流式细胞术提供了一种快速、特异且灵敏的方法来定量体外幽门螺杆菌的粘附。 (C) 1998 Elsevier Science B.V. 保留所有权利。
Adherence may be an important virulence factor for Helicobacter pylori. Current methods available for quantitation of adherence are time consuming and liable to observer error. A new direct technique for fluorescent labelling of bacteria has been developed to quantitate adherence of H. pylori to epithelial cells by fluorescence activated cell sorting (FACS). Type strains of H. pylori, H. mustelae, H. cinaedi and H. fennelliae were grown microaerobically in broth culture for 24 h and fluorescently labelled by incubation with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) at 37 degrees C. After washing to remove excess CFDA-SE, bacteria were co-incubated (ratio 10:1) with gastric epithelial cells at 37 degrees C for up to 24 h. After washing to remove non-adherent bacteria, epithelial cells were detached with EDTA (2 mM) and fixed with formaldehyde for flow cytometry, Adherence was quantitated both in terms of the proportion of cells with adherent H. pylori and as the mean number of adherent bacteria per cell. All H. pylori strains adhered to gastric-type epithelial cells. The proportion of cells with bound bacteria varied from 40-99% and the number of bacteria per cell from 1-50, both of which correlated with microscopy (r = 0.6, and r = 0.8 respectively, n = 35). Time course studies demonstrated saturation of binding by H. pylori within 90 min. For H. mustelae, H. cinaedi and H. fennelliae the proportion of cells with bound bacteria varied from 5-15% and the mean number of bacteria per cell was < 4. Binding of H. pylori to epithelial cells could be partly blocked by pre-incubation with polyclonal anti-sera or using oligosaccharides against potential binding epitopes of gastric mucus. Fluorescent labelling of H. pylori with CFDA-SE in combination with flow cytometry provides a quick, specific, and sensitive method to quantitate in vitro the adherence of H. pylori. (C) 1998 Elsevier Science B.V. All rights reserved.