实验性肝纤维化基质金属蛋白酶-2基因表达与酶活性的变化

Dynamic evolution of MMP-2 gene expression and its enzymatic activities in experimental liver fibrosis

目的探讨肝组织基质金属蛋白酶-2(MMP-2)、金属蛋白酶组织抑制因子-2(TIMP-2) 与肝纤维化发展的关系。方法大鼠随机分成正常对照组、模型组。模型组以二甲基亚硝胺(DMN)腹腔内注射,正常对照组以等渗盐水代替腹腔内注射。分别于第1、4、10、17、28、42、56天分批处死大鼠。留取的肝脏组织做HE与Masson染色,按0-4期标准判定肝纤维化程度,测定羟脯氨酸含量,用半定量逆转录聚合酶链反应检测MMP-2和TIMP-2 mRNA,用酶谱法检测MMP-2的酶活性。结果模型组大鼠肝组织MMP-2 mRNA的表达在动物实验后第10天开始显著高于正常对照组,并保持高水平表达直至动物实验结束;MMP-2活性在动物实验后1d即明显高于正常对照组,并随着实验的进行酶活性增高越明显,在停止DMN损伤后,仍呈高水平表达直至实验结束。TIMP-2 mRNA 17d后表达水平开始下降,到28 d左右时降到最低点,此后表达水平迅速上升,到42 d左右时上升到最高峰,明显高于正常对照水平,保持到动物实验结束。TIMP-2/MMP-2从第10天开始较正常对照明显降低,并随着肝纤维化的发展保持这种显著低于正常对照的水平直至动物实验结束。结论在肝纤维化形成过程中,MMP-2的表达增高,而TIMP-2的表达相对于MMP-2过于低下,从而使MMP-2的酶活性得不到抑制而增高,促进了肝纤维化的不断形成与发展。

Objective To explore the dynamic changes and interactions between MMP-2 and TIMP-2 during experimental liver fibrosis. Methods Wistar rats were randomly allocated into a normal group and a model group. To induce liver fibrosis, rats were injected intraperitoneally with dimethylnitrosamine (DMN) three consecutive times in the first week, then two consecutive times per week, totally for 6 weeks. In the normal control group, rats were injected with saline by the same method as the model group. Animals were sacrificed 1, 4, 10, 17, 28, 42, 56 days after starting DMN injections. Conventional histological examinations of the livers were performed with hematoxylin and eosin and Masson staining. The fibrosis was classified into 0 to 4 stages. Hydroxyproline content was determined after liver tissues were hydrolyzed in HC1 at 160℃ for 2 hrs and then measured with spectrometry at 560nm wavelength. mRNA levels of MMP-2 and TIMP-2 were determined by semi-quantitive RT-PCR. Gelatinase activity of MMP-2 was examined by zymography using gelatin substrate. Results In the model group the hepatic MMP-2 mRNA expression started to increase 10 days after DMN administration and remained at a much higher level than in the normal group throughout the study period, while TIMP-2 mRNA expression started to be lower than in the normal group 17 days after DMN administration and reached the lowest level on the 28th day. Then it rapidly rebounded and remained higher than that in the normal group from the 42nd day to the end of the study period. TIMP-2/MMP-2 began to be lower by several days than that of the normal group after DMN administration through the remaining study period. Zymography showed that the enzymatic activities of both latent MMP-2 and active MMP-2 were increased during the process of liver fibrosis. Conclusion In liver fibrosis, MMP-2 expression increases, while TIMP-2 expression relatively decreases. The enzymatic activities of MMP-2 increase as the liver fibrosis develops.