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目的研究肿瘤坏死因子α(TNFα)对暴发性肝功能衰竭(FHF)小鼠肠黏膜上皮细胞凋亡的作用。方法用D-氨基半乳糖(GalN)+脂多糖(LPS)或TNFα造FHF小鼠模型;酶联免疫吸附法测定血清TNFα含量;光学显微镜和电子显微镜观察肠组织;末端转移酶介导的dUTP缺口末端标记法检测肠组织细胞凋亡情况;免疫组织化学法检测肿瘤坏死因子受体I(TNFRⅠ)蛋白在肠组织的表达与分布。结果实验各组动物各时间点光学显微镜下肠黏膜上皮细胞结构均保持完整,电镜下可见肝功能衰竭组有典型的凋亡细胞;对照组、LPS组、GalN组血清TNFα水平几乎正常,凋亡率低且基本没有TNFRⅠ蛋白表达;GalN+LPS组血清TNFα水平12h明显升高(P<0.01),且TNFRⅠ蛋白在6h(大肠:2.82e+4±4.60e+3;小肠:1.14e+4±2.13e+3)即有表达,此时肠上皮细胞凋亡不明显,9h和12hTNFRⅠ蛋白表达明显增加,同时肠上皮细胞凋亡也明显。用GalN+TNFα造FHF模型,结果也与GalN+LPS组类似。应用抗TNFα抗体,可降低TNFRⅠ蛋白表达和减少肠上皮细胞凋亡的发生。结论TNFα能诱导肝功能衰竭小鼠肠上皮细胞凋亡,抗TNFα抗体能阻断这一作用;在FHF的模型动物中,TNFRⅠ蛋白表达增多,TNFRⅠ蛋白表达与肠上皮细胞凋亡在一定程度上呈正相关。
Objective To investigate the effect of tumor necrosis factor α (TNFα) on apoptosis of intestinal epithelial cells in mice with fulminant hepatic failure (FHF). Methods The FHF mouse model was induced by D-galactosamine (GalN) + lipopolysaccharide (LPS) or TNFα. The content of TNFα in serum was determined by enzyme-linked immunosorbent assay. Intestinal tissue was observed by light microscopy and electron microscopy. The apoptosis of intestinal tissue was detected by nick end labeling method. The expression and distribution of tumor necrosis factor receptor I (TNFRⅠ) protein in intestinal tissue were detected by immunohistochemistry. Results The intestinal mucosal epithelial cells under light microscope were intact and the typical apoptotic cells in liver failure group were observed under electron microscope. The levels of TNFα in control group, LPS group and GalN group were almost normal and apoptosis (P <0.01), and TNFR Ⅰ protein in 6h (large intestine: 2.82e + 4 ± 4.60e + 3; small intestine: 1.14e + 4 ± 2.13e + 3), indicating that the apoptosis of intestinal epithelial cells was not obvious at this time, and the expression of TNFRⅠprotein increased obviously at 9h and 12h, meanwhile the apoptosis of intestinal epithelial cells was also obvious. FHF models were made with GalN + TNFα and the results were also similar to the GalN + LPS group. Anti-TNFα antibody can reduce the expression of TNFR Ⅰ protein and reduce the incidence of intestinal epithelial cell apoptosis. Conclusions TNFα can induce the apoptosis of intestinal epithelial cells in mice with liver failure. Anti-TNFα antibody can block this effect. In FHF model animals, the expression of TNFR Ⅰ protein is increased, the expression of TNFR Ⅰ protein and the apoptosis of intestinal epithelial cells are to a certain extent Was positively correlated.