重组艰难梭状芽孢杆菌毒素TcdB对人体肠上皮细胞层功能的影响

Effects of Recombinant Clostridium di f ficile Toxin B on Function of Human Intestinal Epithelial Monolayer

利用Transwell小室体外建立人结肠细胞T84上皮细胞层,分析不同质量浓度重组艰难梭状芽孢杆菌毒素TcdB(1 000、100、10ng/mL)对上皮细胞层跨膜电阻(TER)的影响;通过免疫荧光染色分析TcdB对上皮细胞层结构的影响;运用荧光标记葡聚糖作为指示剂,检测毒素作用后上皮细胞层的旁通路渗透情况;通过细胞毒性分析,检测上皮细胞层功能受损情况下,TcdB的跨膜渗透。结果发现:重组艰难梭状芽孢杆菌毒素TcdB能引起剂量依赖性单层上皮组织跨膜电阻降低和渗透性增加,在24h内质量浓度为1 000、100、10ng/mL的TcdB能导致上皮细胞层分别丧失100%、70%、50%电阻力,同时分别引起30%、20%、10%的葡聚糖渗透;上皮细胞层受损后,大分子毒素通过旁通路的非正常跨膜渗透也增加。共聚焦分析显示上皮细胞层的细胞间紧密连接结构受损。综上所述,艰难梭状芽孢杆菌毒素TcdB介导的体外培养人T84肠上皮细胞层功能损伤主要表现为电生理学功能异常,旁通路渗透性增加,细胞间紧密连接结构破坏。

Human T84 colonic monolayer electrical resistance （TER） of monolayer was established using transwell chamber. The transepithelial was measured after exposure to recombinant Clostridium difficile TcdB at various concentrations （1 000, 100, 10 ng/mL）. The structure of monolayer was examined by immunofluorescent staining. The permeability of monolayer was monitored by a fluorescence- labeled dextran. The abnormal leakage of TcdB across the damaged monolayer was also detected by acytotoxicity assay. Results showed that recombinant TcdB caused dose-dependent TER decline and permeability of dextran. 1 000, 100 and 10 ng/mL of TcdB resulted in 100%, 70% and 50% loss of TER respectively within 24 h. And the permeable rates of dextran were 30 %, 20 G and 10 %, respectively. An increasing permeability of toxin across the damaged monolayer through the paracellular pathway was also observed. The tight junction structure of monolayer was shown to be disrupted under a confocal microscope. In summary, Clostridium difficile toxin B damages cultured human T84 epithelial monolayer by inducing the electrophysiological dysfunction, increasing the paracellular permeability, and disrupting the tight junction structure.