TNF-α体外对Caco-2细胞通透性的影响及其机制

Tumor necrosis factor-α increases the paracellular permeability of Caco-2 cell monolayers in vitro

目的:探讨肿瘤坏死因子-α(TNF-α)对Caco-2细胞间通透性的影响及作用机制.方法:利用Caco-2细胞建立肠上皮细胞屏障模型,模型分为正常对照组(C组)和TNF-α预处理组(TNF-α组),TNF-α组根据预处理时间又分为3、6、12、24h4个亚组.应用电阻仪测定TNF-α预处理后各组Caco-2细胞的跨上皮细胞电阻(transepithelial electrical resistance,TEER)变化;罗丹明-鬼笔环肽直接染色观察细胞骨架改变;同时采用荧光素酶报告基因检测NF-κB活性.结果:TNF-α各亚组TEER均降低,与C组比较均有显著性差异(均P<0.05),其中TNF-α24h亚组TEER降低明显,与C组及TNF-α3、6、12h亚组比较有显著性差异(均P<0.05);TNF-α3、6、12h亚组NF-κB活性增高,与C组比较均有显著性差异(均P<0.05),且TNF-α12h亚组NF-κB活性明显增高,与C组及TNF-α3、6、24h亚组比较均有显著性差异(均P<0.05);罗丹明-鬼笔环肽直接染色可见C组的F-actin沿细胞膜分布,呈蜂巢状线性荧光,100μg/LTNF-α预处理后导致F-actin荧光信号减弱,并呈锯齿状异常分布.结论:TNF-α导致Caco-2细胞间的通透性增加,其机制可能与NF-κB活化及F-actin重组有关.

AIM： To investigate the effects of tumor necrosis factor-α （TNF-α） on the paracellular permeability of Caco-2 cell monolayers and to explore potential mechanisms involved. METHODS： Caco-2 cells were cultured in vitro to establish a model of intestinal epithelial barrier and divided randomly into two groups： control group and TNF-α treatment group. The TNF-α treatment group was further divided into four subgroups for testing at 3, 6, 12 and 24 h after TNF-α treatment. The changes in transepithelial electrical resistance （TEER） of Caco-2 cells were measured using an electrical resistance system. The changes in cytoskeleton were observed by direct staining with rhodamine-phalloidin. The activation of nuclear factor-κB （NF-κB） was determined by luciferase reporter gene assay. RESULTS： The TEER significantly decreased in all four treatment subgroups compared to the control group （all P 0.05）. The TEER was significantly lower in the 24-h treatment subgroup than in other subgroups （all P 0.05）. The activation level of NF-κB was significantly raised in 3-, 6- and 12-h treatment subgroups compared to the control group （all P 0.05）. The activation level of NF-κB in 12-h treatment subgroup was significantly higher than those in other subgroups （all P 0.05）. In the control group, rhodamine-phalloidin staining showed that F-actin was visualized around the cell membrane and exhibited a honeycomb pattern of fluorescent staining. After treatment with TNF-α, the fluorescence signals were weakened and distributed in a serrated pattern. CONCLUSION： TNF-α increases the paracellular permeability of Caco-2 cell monolayers perhaps by inducing NF-κB activation and actin reorganization.