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目的研究 TNFα对暴发性肝衰竭(FHF)小鼠大肠上皮细胞紧密连接蛋白 occludin 表达的影响。方法采用 D-氨基半乳糖(GalN)和内毒素(LPS)联合腹腔注射(ip)制备 FHF 小鼠动物模型,并设立 TNFα组(TNFα 10μg/kg,ip),TNFα抗体组(注射 LPS 和 GalN 前30min 尾静脉注射TNFα抗体100μg/只)。在不同的时间点(6h,9h)处死动物,应用免疫组织化学技术、Western blot及实时定量 PCR 检测各组小鼠大肠上皮细胞紧密连接蛋白 occludin 表达的变化。结果在生理盐水(NS)对照组,几乎整张切片上均可见到沿大肠黏膜上皮细胞膜顶端呈线性分布的 occludin 阳性染色,但 FHF 组及 TNFα组小鼠的阳性染色较之明显减弱,TNFα抗体组 occludin 的阳性反应与 NS 对照组比较无明显减弱。Western blot 结果与免疫组织化学结果相一致,FHF 组及 TNFα组小鼠9h 时occludin 蛋白含量明显下降,与 NS 对照组相比差异有统计学意义(0.36±0.05,0.48±0.02比0.71±0.09,P<0.05),TNFα抗体组与 NS 对照组相比差异无统计学意义(0.74±0.03比0.71±0.09,P>0.05)。实时定量 PCR 结果显示,FHF 组与 TNFα组小鼠6h 时 occludin mRNA 水平最低,与 NS 对照组相比差异有统计学意义(0.72±0.04,0.81±0.03比1.00±0.05,P<0.05),TNFα抗体组与 NS对照组相比差异无统计学意义(1.01±0.10比1.00±0.05,P>0.05)。结论在小鼠暴发性肝衰竭过程中,TNFα介导大肠上皮细胞间紧密连接蛋白 occludin 表达的下降。
Objective To investigate the effect of TNFα on the expression of tight junction protein occludin in large intestinal epithelial cells in rats with fulminant hepatic failure (FHF). Methods FHF mice were induced by GalN, LPS and intraperitoneal injection (ip). TNFα (TNFα 10 μg / kg, ip), TNFα antibody group (injected with LPS and GalN The first 30min tail vein injection of TNFα antibody 100μg / only). The animals were sacrificed at different time points (6h, 9h). Immunohistochemistry, Western blot and real-time quantitative PCR were used to detect the expression of occludin in colorectal epithelium. Results Oral occludin positive staining along the top of membrane of large intestinal mucosa was observed in NS group and almost in whole section. However, the positive staining of FZF group and TNFα group mice was significantly weaker than that of normal group. TNFα antibody Occludin positive reaction group and NS control group no significant reduction. Western blot results were consistent with the results of immunohistochemistry, occludin protein levels in FHF group and TNFα group at 9h were significantly decreased compared with NS control group (0.36 ± 0.05,0.48 ± 0.02 vs. 0.71 ± 0.09, P <0.05). There was no significant difference between TNFα antibody group and NS control group (0.74 ± 0.03 vs 0.71 ± 0.09, P> 0.05). The results of real-time PCR showed that occludin mRNA level was the lowest in FHF group and TNFα group at 6h, which was significantly lower than NS control group (0.72 ± 0.04,0.81 ± 0.03 vs 1.00 ± 0.05, P <0.05) There was no significant difference between antibody group and NS control group (1.01 ± 0.10 vs. 1.00 ± 0.05, P> 0.05). Conclusion In the process of fulminant hepatic failure, TNFα mediates the decrease of occludin expression in large intestine epithelial cells.