摘要
目的旨在探讨人前梯度蛋白2(anterior gradient-2,AGR2)在肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导的肠黏膜屏障损伤中的作用及其机制。方法通过体外培养人结肠腺癌细胞株Caco-2细胞建立肠黏膜屏障模型,预先转染AGR2质粒和对照质粒,24 h后实验组加入TNF-α刺激细胞,通过实时定量PCR及Western blot检测AGR2表达,通过跨膜电阻检测细胞膜通透性,透射电镜下观察线粒体自噬的发生。结果Caco-2细胞培养21 d,跨膜电阻值(transepithelial electrical resistance,TEER)大于600Ω·cm2,趋于稳定,证实体外肠黏膜屏障模型成功构建。在TNF-α诱导的肠上皮屏障损伤模型中,AGR2 mRNA及蛋白表达明显下调,TEER下降至200Ω·cm2左右,透射电镜下显示线粒体形态破坏。而预先转染AGR2质粒可抑制TNF-α引起的跨膜电阻值下降,TEER维持在400Ω·cm2左右,透射电镜下可见线粒体自噬小体产生。结论体外实验证实AGR2基因可抑制TNF-α诱导的肠黏膜屏障损伤,保护线粒体功能,这种保护作用可能是通过诱导线粒体自噬发生实现的。
Objective To investigate the role and mechanism of anterior gradient-2(AGR2)in intestinal mucosal barrier injury induced by tumor necrosis factor-α(TNF-α).Methods Caco-2 cell monolayers were pre-transfected with an AGR2 plasmid and then exposed to TNF-α.Real-time PCR was employed to detect AGR2 mRNA expression,with western blot detection of AGR2 protein expression.Epithelial permeability was assessed by detecting transepithelial electrical resistance.Electron microscope was used to observe autophagic bodies.Results Both AGR2 mRNA and protein expression levels were significantly reduced by TNF-αexposure compared with the levels in untreated control monolayers.AGR2 overexpression signifcantly ameliorated TNF-αinduced epithelial barrier hyperpermeability,and mediated mitophagy in Caco-2 cell monolayers.Conclusion This study suggested that AGR2 could inhibit TNF-αinduced intestinal barrier dysfunction and this protective mechanism might be promoted by mediated mitophagy in Caco-2 cell monolayers.
作者
叶晓琳
吴捷
赵志强
孙梅
Ye Xiaolin;Wu Jie;Zhao Zhiqiang;Sun Mei(Department of Pediatrics,Shengjing Hospital of China Medical University,Shenyang 110004,China;Mudanjiang City,Heilongjiang Province Forestry Center Hospital,Mudanjiang 157000,China)
出处
《国际儿科学杂志》
2020年第3期208-212,共5页
International Journal of Pediatrics
