肝纤维化过程中胶原、基质金属蛋白酶及其抑制因子表达的动态变化及相互关系

Dynamic evolution of MMP-13, TIMP-1, type Ⅰ and Ⅲ collagen and their interaction in experimental liver fibrosis

目的 研究在整个肝纤维化形成过程中肝组织Ⅰ、Ⅲ型胶原,间质性胶原酶(MMP-13)及其抑制因子(TIMP-1)表达水平的动态变化规律及相互间的关系。 方法 将大鼠随机分成正常对照组与模型组。模型组以二甲基亚硝胺腹腔内注射,第1周注射3次,随后每周2次,共注射6周,停用后再观察2周;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,应用半定量RT-PCR检测Ⅰ、Ⅲ型胶原、MMP-13和TIMP-1 mRNA。 结果 在肝纤维化形成过程中,胶原持续增高,其中Ⅰ型胶原mRNA在肝组织受损后10 d开始较正常对照明显增高(正常组0.468±0.015,模型组0.603±0.031,t=2.85,P<0.05),并保持不断增高的表达水平直至实验结束;Ⅲ型胶原mRNA从28 d左右开始显著增高(正常组0.774±0.043,模型组0.922±0.079,t=4.16,P<0.01),直至实验结束;TIMP-1 mRNA从第4天即开始明显增高(正常组0.618±0.030,模型组0.728±0.013,t=2.60,P<0.05),并保持不断增高的趋势直至实验结束;MMP-13 mRNA从10 d后到28 d有显著增高(17 d左右达到高峰,正常组0.987±0.048,模型组1.141±0.033,t=4.08,P<0.01),此后便逐渐下降至实验结束。 结论 在肝纤维化?

Objective To obtain a detailed pattern of the dynamic evolution and interactions among MMP-13, TIMP-1, type I and Ⅲ collagen during experimental liver fibrosis. Methods Wistar rats were randomly allocated into a normal group, and a model group. To induce liver fibrosis, rats were intraperitoneally injected 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 treated with saline by the same means. Animals were sacrificed 1, 4, 10, 17, 28, 42, 56 days after starting DMN injections. Coventional histological examinations were performed after hematoxylin and eosin, and Masson stain. Fibrosis stages were classified into 0 to 4. Hydroxyproline contents were determined after liver tissues were hydrolyzed in HG1 at 160℃ for 2 h and then measured with spectrometry at 560 nm wavelength. mRNA levels of MMP 13, TIMP-1, type I and Ⅲ collagen were determined by semi-quantitive RT-PCR. Results In the model group, hepatic type I pro-collagen mRNA expression started to increase on the 10th day after DMN administration (t = 2.85, P < 0.05), type Ⅲ started to increase on the 28th day (t = 4.16, P < 0.01), and TIMP-1 mRNA expression started to increase on the 4th day (t = 2.60, P < 0.05). They all remained much higher than in the normal group throughout the remaining study period. Hepatic MMP-13 mRNA expression started to increase on the 17th day after DMN administration and remained at a higher level than in the normal group until he 28th day (t = 4.08, P < 0.01), then gradully returned to normal level at the end of the study period. Conclusion Although hepatic MMP-13 expression transiently increased during liver fibrosis, enhanced expression of TIMP-1 from the early periods of liver fibrosis inhibited the collagen degrading ability of MMP-13, therefore, over-expressed collagen accumulated in the liver. Thus, it is hypothesized that TIMPs play a pivotal role in liver fibrosis.