摘要
目的探讨乙醇引起肠黏膜屏障的破坏以及这种破坏机制是否与紧密连接蛋白ZO-1相关。方法培养人结肠腺癌细胞株Caco-2,分别设正常对照组:不加刺激物及干预因素;实验组:加入不同体积分数(1%、2.5%、5%、7.5%、10%)乙醇分别刺激不同时间(0～3h),四甲基偶氮唑盐(MTT)比色法检测细胞生存率;测定跨上皮电阻(TEER)和荧光黄的透过量反映肠上皮细胞单层通透性;选定体积分数5%乙醇作为实验浓度,应用蛋白印迹法检测紧密连接蛋白ZO-1表达的动态变化。结果体积分数为5%的乙醇未影响到细胞的生存率。不同浓度乙醇作用20min后,细胞单层通透性增加,60min达高峰,TEER下降,荧光黄透过增加,以体积分数5%乙醇最明显。蛋白印迹法检测体积分数5%乙醇在1h内致Caco-2细胞表达ZO-1减少。结论乙醇可引起肠上皮黏膜屏障破坏,其机制可能和紧密连接蛋白ZO-1的破坏相关。
Objective To explore whether ethanol can disrupt the intestinal epithelial barrier and whether it is associated tight junction protein ZO-1. Methods Caco-2 cells were cultured with DMEM with 4.5 mg/mL glucose, 50 U/mL penicillin, 50 U/mL streptomycin, and 10 % FBS. Cells were then treated with ethanol （1%, 2.5 %, 5 %, 7.5 %, 10%）for 0-3 h, MTT was used to detect cell vigor. Epithelial monolayer permeability was measured by transepithelial electrical resistance and mucosal to serosal flux of paracellular marker luminal yellow. 5 % etha- nol was elected at last. Western blotting was used to detect expression of ZO-1. Results 50//00 ethanol had no effect on Caco-2＇s vigor. Different concentrations of ethanol increased the epithelial paracellular permeability. TEER decreased and luminal yellow flux increased. 5% ethanol was the most obvious. ZO-1 was decreased with in 1 h by Western blotting in 5% ethanol group. Conclusion Ethanol disrupts the intestinal epithelial barrier. Its mechanism may be correlated to the decreased expression of ZO-1 protein.
基金
辽宁省科技厅高等学校科研项目(2009A809)
