Establishment of Caco-2 cell monolayer model and barrier permeability

Abstract

Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability. Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts.The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance(TEER)and lucifer yellow flux, and verified by transmission electron microscope.Different concentrations of PAF(0, 50, 100, and 200 nmol/L)were exposed for 24 hours to Caco-2 monolayer when cultured 21 days.The tight junction was observed under transmission electron microscope.Assessment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analysis. Results Cultured Caco-2 cell confluencd as monolayer with time passed.From 5th day, TEER increased, then reached 600Ω·cm2 at 15th day and lasted to 21st day, there was little flux of lucifer yellow, transmission electron microscopy also found cells differentiated better, had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro.TEER decreased and lucifer yellow flux increased in cells exposed to PAF.The permeability reached the peak when exposed to 100 nmol/L PAF(P< 0.01), tight junction disrupted, ZO-1 protein expression downregulated, abnormal localization and distribution was assessed by immunofluorescence staining. Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.PAF increased intestinal epithelial permeability, which would correlate to the decreased protein expression and abnormal distribution of ZO-1. Key words: Human colon carcinoma cell line(Caco-2); Permeability; Transepithelial electrical resistance; Platelet-activating factor(PAF); ZO-1