University of Maryland, Mucosal Biology Research Center, Baltimore, Maryland, USA.
Infect Immun. 2013 Mar;81(3):876-83. doi: 10.1128/IAI.01406-12. Epub 2013 Jan 7.
Helicobacter pylori infection of the stomach is related to the development of diverse gastric pathologies. The ability of H. pylori to compromise epithelial junctional complexes and to induce proinflammatory cytokines is believed to contribute to pathogenesis. The purpose of this study was to use an in vitro human gastric epithelial model to investigate the ability of H. pylori to affect permeability and the extent and polarity of the host inflammatory response. NCI-N87 monolayers were cocultured with live or heat-killed H. pylori or culture supernatants. Epithelial barrier function was measured by transepithelial electric resistance (TEER) analysis, diffusion of fluorescein isothiocyanate (FITC)-labeled markers, and immunostaining for tight junction proteins. Supernatants from both apical and basolateral chambers were tested for cytokine production by multiplex analysis. H. pylori caused a significant decrease in TEER, an increased passage of markers through the infected monolayer, and severe disruption and mislocalization of ZO-1 and claudin-1 proteins. Cell viability was not altered by H. pylori, indicating that loss of barrier function could be attributed to a breakdown of tight junction integrity. Significantly high levels of cytokine secretion were induced by either viable or heat-killed H. pylori. H. pylori affects monolayer permeability of polarized human gastric epithelial cells. Proinflammatory cytokines were secreted in a polarized manner, mostly basolaterally. Live bacteria are required for disruption of tight junctions but not for the induction of cytokine secretion. The NCI-N87 cell line provides an excellent model for the in vitro study of H. pylori pathogenesis and the epithelial cell host response to infection.
幽门螺杆菌感染胃部与多种胃部病理变化有关。幽门螺杆菌破坏上皮细胞连接复合体并诱导促炎细胞因子的能力被认为是导致发病的原因。本研究的目的是使用体外人类胃上皮细胞模型来研究幽门螺杆菌影响通透性以及宿主炎症反应的程度和极性的能力。NCI-N87 单层细胞与活的或热杀死的幽门螺杆菌或培养上清液共培养。通过跨上皮电阻 (TEER) 分析、荧光素异硫氰酸酯 (FITC) 标记物的扩散和紧密连接蛋白的免疫染色来测量上皮屏障功能。通过多重分析测试来自顶端和基底外侧腔室的上清液中细胞因子的产生。幽门螺杆菌导致 TEER 显著降低，标记物通过感染单层的通透性增加，以及 ZO-1 和 Claudin-1 蛋白的严重破坏和定位错误。幽门螺杆菌不会改变细胞活力，表明屏障功能的丧失可能归因于紧密连接完整性的破坏。活的或热杀死的幽门螺杆菌均可诱导显著高水平的细胞因子分泌。幽门螺杆菌影响极化人胃上皮细胞单层的通透性。促炎细胞因子以极化方式分泌，主要是基底外侧。破坏紧密连接需要活细菌，但不需要诱导细胞因子分泌。NCI-N87 细胞系为体外研究幽门螺杆菌发病机制和上皮细胞对感染的宿主反应提供了极好的模型。
