酒精性肝病细胞凋亡的相关机制研究

Correlation mechanism of alcoholic liver disease apoptotis

目的探讨大鼠酒精性肝病细胞凋亡与细胞色素P4502E1、TNF-α及氧自由基的关系。方法采用灌胃法制备大鼠酒精性肝病(ALD)模型,模型组用40%酒精8g/(kg·d)分2次灌胃,共8周,对照组灌等量的生理盐水。采用TUNEL法检测肝细胞的凋亡、用PCR法测定细胞色素P4502E1的表达、放射免疫法检测血清TNF-α含量、硫代巴比妥酸(TBA)法测血清丙二醛(MDA)的含量、黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)的活力。结果模型组凋亡的肝细胞明显增多,主要分布在中央静脉周围、点状和灶状坏死区。CYP2E1表达:对照组c1基因频率为91.65%、c2基因频率为8.35%;模型组c1基因频率为53.35%、c2基因频率为46.65%,差异均有显著性(P<0.05)。血清TNF-α水平与肝细胞凋亡指数及SOD与MDA水平之间有相关性(TNF:AI,r=+0.836;SOD:MDA,r=-0.582)。结论长期酒精摄入可引起肝细胞凋亡增多,CYP2E1基因PstI及RsaIRFLPs与酒精性肝病有关,其中c2基因可能与大鼠酒精性肝病的发生有关,TNF-α和氧自由基及脂质过氧化损伤在酒精性肝病的肝细胞凋亡过程中起一定作用。

[Objective] To investigate the relationship between the hepatocyte apoptosis, CYP2E1, TNF-α and oxygen free radical in alcoholic liver disease（ALD）in rats. [Methods] 70 rats were randomized into two groups, first group was administered 40% ethanol [8 g/（kg· d）], intragastrically by gavage twice daily in ALD model rats for 8 weeks, the control rats received same volume of saline by gavage. The hepatocyte apoptosis was detected by the TUNEL method, expression of serum CYP2E1 was determined by polymerase chain reaction（PCR） method, the TNF levels of serum were determined by radio immunoassay method, the malonaldehyde （MDA） was determined by thibabitufic acid（TBA） quantifying method. The erythrocuprein（SOD） was determined by thibabitufic pufine oxidase method. [Results] The TUNEL positive cells were located around the central vein, spotty and focal necrosis in the ALD group. The apoptotic index（Al） in the ALD group was significantly higher than that in the control group （P 〈 0.05）. Expression in CYP2E1： the allelic ftequency of el and c2 were 91.65%, and 8.35% respectively, in cotrol group. The allelic frequency of el and c2 were 53.35% and 46.65%, respectively, in ALD group, there were significantly differences between two groups （all P 〈0.05）. The level of serum tumor necrosis factor-α（TNF-α）and maleic dialdehyde （MDA） in the ALD group were significantly higher than those in the control group （P 〈0.05）, the serum SOD then decreased （P 〈0.05）. [Conclusion] Chronic alcohol administration induced alcoholic liver disease and liver dysfunction, and increased hepatocyte apoptosis. Rsa I and Pst I RFLPs related with ALD in model rats, and c2 gene might be related to development of ALD. The TNF-α, oxygen free radicals and lipid peroxidation reaction have certain effect in hepatocyte apoptosis of ALD process.