鼠暴发性肝衰竭中Fas表达与肝细胞凋亡的关系

Relationship between hepatocytic apoptosis and Fas expression in mouse fulminant hepatic failure

目的:研究Fas表达及肿瘤坏死因子-α(tumor necrosis factor- α,TNF-α)在暴发性肝衰竭(fulminant hepatic failure, FHF)中与肝细胞凋亡的关系. 方法:采用脂多糖(LPS)和D-氨基半乳糖(D-GAIN)联合用药制备FHF小鼠模型;采用免疫组化方法检测肝组织Fas表达, 分别采用ELISA方法和RT-PCR法检测血清TNF-α水平及肝组织TNF-αmRNA表达;采用肝组织DNA琼脂糖凝胶电泳和TUNEL法检测肝细胞凋亡;在用药后2,4,8,12 h 的不同时期动态观察Fas表达、血清TNF-α水平及肝组织TNF-αmRNA表达及肝细胞凋亡的变化,并对模型鼠给予TNF-αmAb,动态观察上述指标的变化. 结果:在FHF模型小鼠中,用药后2 h开始Fas有少量表达,至8 h和12 h表达均很多,二者比较无显著差异, 与2 h组比较P<0.01,与4 h组比较P<0.05.用药后2-4 h 肝组织TNF-αmRNA表达显著增加(0.91±0.75,正常值为0.32±0.10),伴血清TNF-α水平升高(320±87 ng/L,正常值为17±7 ng/L),8h可出现典型的肝细胞凋亡表现,血清ALT和TBil水平显著增加(分别为9352±1 000nkat/L和163.7±34.5 μmoL/L,正常值分别为393±134 nkat/L 和14.9±4.8 μmoL/L),12 h肝细胞坏死和凋亡同时存在, 血清ALT和TBil水平达最高峰(分别为11141±1312nkat/L 和203.2±19.9 μmoL/L).给予TNF-αmAb后LPS/D-GalN 介导的肝细胞凋亡和损伤被阻断,Fas表达亦被阻断. 结论:在FHF中,TNF-α对肝细胞凋亡及肝损伤起重要的作用,肝细胞凋亡的发生与Fas的表达增加有关.

AIM: To study the relationship among tumor necrosis factor-α, Fas expression and hepatocyte apoptosis in an experimental model of fulminant hepatic failure (FHF). METHODS: A mouse model of FHF was established by LPS and D-GalN. The expression of Fas in liver tissue was detected by immunohistochemical method. Serum TNF-α level and TNF-α mRNA expression in liver were analyzed by ELISA and RT-PCR method respectively. Hepatocytic apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNF-α, Fas and hepatocytic apoptosis were observed in the different stage after drug administration. In addition, changes of the above items were observed after pretreatment with anti-TNF-αIgG1. RESULTS: There was a little expression of Fas at 2 h in model group. The expression of Fas increased distinctly at 8 h and 12 h and there was no statistical difference between them. The expression of Fas at 8 h and 12 h was higher than that at 2 h and 4 h (P<0.01 and P<0.05, respectively). TNF-α mRNA expression in liver increased statistically (0.91±0.75) and the data of normal control was (0.32±0.10) in 2 hours to 4 hours after administration of LPS and D-GalN. The level of serum TNF-α increased (320±87 ng/L, the data of normal control was 17±7 ng/L). There was typical manifestation of hepatocytic apoptosis at 8 h after the drug administration. The level of serum ALT and TBil obviously increased (9 352±1 000 nkat/L and 163.7±34.5 μmoL/L, respectively, the data of normal control was 393±134 nkat/L and 14.9±4.8 μmoL/L, respectively). and there were hepatocytic apoptosis and necrosis at 12 hour after drug administration, at the same time, the level of serum ALT and TBil reached the peak (11 141±1 312 nkat /L and 203.2±19.9 μmol/L, respectively). Hepatocytic apoptosis and liver injury and the expression of Fas could be blocked after antagonized with TNF-α. CONCLUSION: TNF-α plays an important role on hepatocydc apoptosis and liver injury in fulminant hepatic failure. The hepatocytic apoptosis induced by TNF-α is correlated with the expression of Fas.