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目的探讨乙醇对肠上皮细胞屏障功能的影响及作用机制。方法将不同浓度的乙醇与肠上皮细胞株Caco-2细胞共培养4h,采用四甲基偶氮唑盐(MTT)比色法及乳酸脱氢酶法(LDH)检测细胞存活率。应用Caco-2细胞建立肠上皮细胞屏障模型,不同浓度乙醇溶液处理后,测定其跨上皮细胞电阻值(TEER)及荧光黄透过率的变化。应用蛋白印迹法及免疫荧光染色法检测紧密连接蛋白occludin的表达和分布。结果乙醇浓度≤5%时,不影响Caco-2细胞的生存率。乙醇诱导肠上皮细胞屏障通透性增加,呈浓度-时间依赖性;乙醇作用后,细胞膜上occludin表达逐渐降低,60min时达最低值,细胞膜上呈点状分布,此后部分可逐渐恢复,趋势与值及荧光黄透过率一致。结论乙醇可破坏细胞间紧密连接蛋白,进而诱导肠上皮细胞屏障通透性增加。
Objective To investigate the effect of ethanol on the barrier function of intestinal epithelial cells and its mechanism. Methods Different concentrations of ethanol were co-cultured with intestinal epithelial cell line Caco-2 for 4 hours. Cell viability was determined by MTT assay and lactate dehydrogenase (LDH) assay. The intestinal epithelial cell barrier model was established by using Caco-2 cells. After treated with different concentrations of ethanol solution, the trans-epithelial cell resistance (TEER) and fluorescence yellow transmittance were measured. Western blotting and immunofluorescence staining were used to detect the expression and distribution of tight junction protein occludin. Results The ethanol concentration of 5% did not affect the survival rate of Caco-2 cells. Ethanol induced an increase in the barrier permeability of intestinal epithelial cells in a time-and concentration-dependent manner. After ethanol treatment, occludin expression was gradually decreased at 60 min and reached a minimum at 60 min. Value and fluorescence yellow transmittance. Conclusion Ethanol can disrupt the tight junction protein between cells and induce the increase of barrier permeability of intestinal epithelial cells.