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目的:探讨肿瘤坏死因子(TNF-α)对肠黏膜上皮细胞间紧密连接的影响. 方法:应用免疫荧光方法检测在肿瘤坏死因子(TNF-α)0, 50,100和200μg/L作用下,与结肠癌上皮细胞株(CaCo- 2细胞株)共同培养24 h,以及用TNF-α100 μg/L与CaCo- 2细胞共同培养0,4,8和24 h,观察TNF一-α对紧密连接蛋白(zonula occluden 1,ZO-1)表达的影响.并应用半定量RT-PCR方法检测TNF-α用上述相应的时间和浓度作用后对肠黏膜上皮细胞ZO-1 mRNA表达的影响. 结果:用TNF-α100和200 μg/L作用24 h后ZO-1的免疫荧光明显减少,并呈剂量依赖性;用TNF-α100 μg/L作用24 h时ZO-1免疫荧光明显减少,并呈时间依赖性.应用半定量RT-PCR的方法检测结果:TNF-α作用24 h时, ZO-1 mRNA从TNF-α0μg/L的1.1926降至100μg/L的0.7834和200 μg/L的0.7081;TNF-α100μg/L作用4 h, ZO-1mRNA为(95.5±5.5%);8 h为(82.0±5.4%);24 h 降至最低为(67.7±5.7%),与对照组相比P<0.01. 结论:TNF-α是通过抑制肠上皮细胞间紧密连接蛋白ZO-1 表达引起肠黏膜上皮细胞间紧密连接破坏.并且是通过抑制ZO-1 mRNA引起肠上皮细胞ZO-1蛋白表达降低.
Objective: To investigate the effect of tumor necrosis factor-α (TNF-α) on the intestinal mucosal epithelial tight junctions.Methods: Immunofluorescence was used to detect the expression of tumor necrosis factor alpha (TNF-α) (CaCo-2 cell line) for 24 h, and co-cultured with CaCO-2 cells at a concentration of 100 μg / L TNF-α for 0, 4, 8 and 24 h. The effect of TNF-α on the expression of tight junction protein zonula occluden 1, ZO-1) expression.METHODS: The effect of TNF-α on the expression of ZO-1 mRNA in intestinal mucosa epithelial cells was detected by semi-quantitative RT-PCR.Results: The immunofluorescence of ZO-1 was significantly reduced in a dose-dependent manner at-100 and 200 μg / L for 24 h. The immunofluorescence of ZO-1 at 100 μg / L TNF-α was significantly decreased in a time-dependent manner The results of semi-quantitative RT-PCR assay showed that ZO-1 mRNA decreased from 1.1926 at TNF-α0μg / L to 0.7834 at 100μg / L and 0.7081 at 200μg / L at 24 hours after treatment with TNF-α; / L for 4 h, ZO-1 mRNA was (95.5 ± 5.5)% for 8 h, (82.0 ± 5.4%) for 8 h and (67.7 ± 5.7% : TNF-α is by inhibition Epithelial tight junction protein ZO-1 expression caused by intestinal epithelial tight junction destruction. And ZO-1 by inhibition of the mRNA cause intestinal epithelial cells decreased ZO-1 protein expression.