Abstract:
Human T84 colonic monolayer was established using transwell chamber. The transepithelial electrical resistance (TER) of monolayer 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 a cytotoxicity 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% 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.