Logan R P, Robins A, Turner G A, Cockayne A, Borriello S P, Hawkey C J
Division of Gastroenterology, University Hospital, Nottingham, UK.
J Immunol Methods. 1998 Apr 1;213(1):19-30. doi: 10.1016/s0022-1759(98)00018-0.
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.
黏附可能是幽门螺杆菌的一个重要毒力因子。目前可用的定量黏附的方法耗时且容易出现观察者误差。已开发出一种用于细菌荧光标记的新直接技术，通过荧光激活细胞分选（FACS）来定量幽门螺杆菌对上皮细胞的黏附。幽门螺杆菌、鼬形螺杆菌、西奈螺杆菌和芬内利螺杆菌的标准菌株在肉汤培养中微需氧培养24小时，并在37℃下与羧基荧光素二乙酸琥珀酰亚胺酯（CFDA-SE）孵育进行荧光标记。洗涤以去除过量的CFDA-SE后，细菌在37℃下与胃上皮细胞共孵育（比例为10:1）长达24小时。洗涤以去除未黏附的细菌后，用EDTA（2 mM）使上皮细胞脱离并用甲醛固定以进行流式细胞术分析。黏附通过黏附幽门螺杆菌的细胞比例以及每个细胞黏附细菌的平均数来定量。所有幽门螺杆菌菌株均黏附于胃型上皮细胞。有结合细菌的细胞比例在40%-99%之间，每个细胞的细菌数在1-50个之间，这两者均与显微镜检查相关（分别为r = 0.6和r = 0.8，n = 35）。时间进程研究表明幽门螺杆菌在90分钟内结合达到饱和。对于鼬形螺杆菌、西奈螺杆菌和芬内利螺杆菌，有结合细菌的细胞比例在5%-15%之间，每个细胞的细菌平均数<4。幽门螺杆菌与上皮细胞的结合可通过与多克隆抗血清预孵育或使用针对胃黏液潜在结合表位的寡糖部分阻断。用CFDA-SE对幽门螺杆菌进行荧光标记并结合流式细胞术提供了一种快速、特异且灵敏的方法来体外定量幽门螺杆菌的黏附。
