基质金属蛋白酶抑制剂-1在实验性酒精性肝病中的动态表达

Dynamic expression of tissue inhibitor of metalloproteinase-1 in alcoholic liver disease in rats

目的探讨基质金属蛋白酶抑制剂1(TIMP-1)在大鼠酒精性肝病发生发展中的动态变化及表达。方法应用灌胃的方法制备大鼠酒精性肝病的动物模型,肝组织作HE染色及Masson三色胶原染色,Masson染色结果用病理图象分析仪分析,测定胶原纤维面积百分比。应用免疫组织化学技术检测TIMP-1在酒精性肝病中的表达,其结果用病理图象分析仪分析。应用流式细胞术检测酒精性肝病不同阶段TIMP-1 FI值的动态变化。结果肝脏组织病理学改变,对照组HE染色可见正常中央静脉及放射状排列的肝板。模型组4wk末肝细胞轻度脂胁变性及点状坏死。8wk末肝细胞中重度脂肪变性,炎性细胞浸润加重,Masson染色终末静脉周围胶原纤维增因加。12wk末及16wk末肝细胞坏死增多,炎性细胞浸润成团,Masson染色终末静脉增厚,胶原纤维向窦周隙延伸,窦周纤维组织较前增加,造模成功。免疫组化研究表明,TIMP-1主要位于血管内皮细胞、窦内皮细胞、肝窦细胞及静脉周围肝细胞的胞质。病理图象分析结果示对照组4,8,12,16wk TIMP-1面积比分别为4.8±0.7,4.8±0.6,5.2±0.8,5.1±0.7,模型组四个阶段分别为9.8±1.4,16.7±2.4,21.3±3.4,25.3±3.7,明显高于对照组(P<0.05)。流式细胞术检测示对照组TIMP-1 FI值四个阶段分别为0.4±0.1,0.5±0.2,0.4±0.1,0.5±0.1,模型组分别为0.6±0.1,0.8±0.1,1.0±0.1,1.2±0.2,明显高于对照组(P<0.05),并且随造模时间延长其含量进行性增加。结论在大鼠酒精性肝病发生发展中,TIMP-1水平进行性增加,提示其可能参与酒精性肝病的进展。

AIM To evaluate the dynamic changes and expression of tissue inhibitor of metalloproteinase-Ⅰ (TIMP-1) in alcoholic liver disease in rats. METHODS Alcoholic liver disease model in rats was established by gavage. Masson trichome was used to detect the expression of collagen. The expression of TIMP-Ⅰ was studied by immunohistochemistry and the results were analyzed by image analysis system. Flow cytometry was used to study the dynamic change of fluorescent index (FI) of TIMP-1 in the different stages of alcoholic liver disease in rats. RESULTS By HE staining, normal central vein and normal hepatic plates were shown in control group. In alcoholic liver disease model at the end of 4 weeks, there were light steatosis and spotty hepatocellular necrosis. At the end of 8 weeks, there was moderate or severe steatosis and a lump of inflammatory cells, and the quantities of collagenous fibers around terminal vein increased by Masson trichome. At the end of 12 and 16 weeks, we could see local hepatocellular necrosis and increased inflammatory cells by HE stain, The collagen of terminal vein was thickened, and collagenous fibers spread into perisinusoidal areas. Moreover, the collagen of perisinusoidal areas increased by Masson trichome and alcoholic liver disease model was established successfully. By immunohistochemistry, immunoreactive TIMP-1 was expressed in endothelial cells of hepatic artery and portal vein, sinusoidal endothelial cells and sinusoidal cells. It was also expressed in the cytoplasm of cells around vein. According to image analysis, the level of TIMP-1 was higher in rats of model group (9.8±1.4,16.7±2.4, 21.3±3.4 and 25.3±3.7 at 4, 8, 12 and 16 weeks respectively) than in those of control group (4.8±0.7, 4.8±0.6, 5.2±0.8 and 5.1±0.7 at 4, 8, 12 and 16 weeks, P<0.05). By flow cytometry, the expression of TIMP-1 was higher in rats of model group (0.6±0.1, 0.8±0.1, 1.0±0.1 and 1.2±0.2 at 4, 8, 12 and 16 weeks) than in those of control group (0.4±0.1, 0.5±0.2, 0.4±0.1 and 0.5±0.1 at 4.8, 12 and 16 weeks, P<0.05). The level of TIMP-1 increased gradually with the progress of alcoholic liver disease. CONCLUSION TIMP-1 increased with the progress of alcoholic liver disease, which might be involved in the development of alcoholic liver disease.