摘要
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。
Objeαive We reported recently that intestinal epithelial tight junαion protein occludin was reduced in mice with fulminant hepatic failure （ FHF）, when there was accompanying high levels of serum TNFα. This study was to explore the role of TNFα in the changes of intestinal epithelial tight junαion protein occludin in mice with FHF. Methods Male BALB/c mice were divided into 6 groups. In one group, the mice were intraperitoneally injeαed with lipopolysaccharide （LPS, 10 i.~g/kg ） and D- galaαosamine （GAIN, 800 mg/kg） to induce acute liver necrosis. Anti-TNFα-IgG（ 100 trig/mouse） was injeαed through vena caudalis 30 minutes before GalN/LPS administration. The remaining groups were intraperitoneally injeαed with GAIN, LPS, TNFα（ 10 μg/kg） or saline respeαively to serve as controls. Mice were executed 6 and 9 hours after administration and intestinal specimens were obtained. The location and expression of intestinal epithelial tight junαion protein occludin were deteαed with immunohistochemistry and Western blot. Real time quantitative PCR was used to deteα the mRNA level of occludin. Results Tight junαion protein occludin was localized on the apical region of lateral plasma membrane representing the region of tight junαions in the surface and crypt epithelial cells. In the mucosal tissue from mice with FHF and with TNFα injeαion for 9 hours, occludin-staining was globally down-regulated. Results from Western blot demonstrated consistent and significant reduαion of occludin expression in the mice with FHF （0. 36± 0.05 vs 0.71 ±0.09, P 〈0.05） and with TNFα injeαion for9 hours （0.48 ±0.02 vs 0.71±0.09, P〈 0. 05）. Anti-TNFα-IgG can prevent the changes of occludins as examined both with immunohistochemistry and with Western blot （0.74 ±0. 03 vs 0. 71± 0. 09, P 〉 0. 05 ） . Furthemore we evaluated occludin mRN A expression by real time quantitative PCR and observed marked down-regulation of occludin mRNA in the mice with FHF and TNFα injeαion for 6 hours
出处
《中华内科杂志》
CAS
CSCD
北大核心
2007年第6期478-481,共4页
Chinese Journal of Internal Medicine
基金
国家自然科学基金(30670947)
