霍乱弧菌致病因子诱导人肠上皮细胞炎症反应的实验研究

Identification of the secreted component(s) of V.cholerae that induces an intestinal epithelial inflammatory response

目的:鉴定诱导人肠上皮细胞炎性反应的霍乱弧菌分泌产物。方法:野生型霍乱弧菌O1株C6706及其变异株培养上清刺激极化的人单层肠上皮T84细胞体外模型上层腔面,分析细胞受刺激后通透性和细胞活性以及白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)基因表达改变;分析上清中霍乱弧菌蛋白酶(Vibrio choleraeprotease,PrtV)和霍乱弧菌溶细胞素(Vibrio choleracytolysin,VCC)含量以及溶血能力。结果:PrtV缺失株培养上清可引起T84细胞层的炎性反应,表现为通透性增加和IL-8,TNF-α基因表达增加。肠上皮细胞炎性反应及红细胞溶血反应与VCC活性有关。结论:VCC是霍乱弧菌引起人肠上皮细胞炎性反应的主要因素,PrtV通过降解VCC而降低对肠上皮细胞的炎性反应。

Objective： To identify the secreted component （s） of V. cholerae that induces an intestinal epithelial inflammatory response. Methods： Culture supernatants of wild type Vibrio cholerae O1 strain C6706 and its derivates were analyzed for capacity to induce changes in cytokine mRNA expression levels, IL-8 and TNF-α secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supematants were also analyzed for PrtV and VCC content by immunoblot analyses and for hemolytic capacity. Results： Experiments with culture supernatants of different V. cholerae derivates suggested that VCC was capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-α in tight monolayers. Capacity to induce this response in epithelial ceils correlated to VCC content and hemolytic activity. Conclusion： The findings suggested that VCC induced the human intestinal epithelial inflammatory response. Furthermore, PrtV can mediate an environment dependent modulation of VCC induced inflammatory response.