CCl_4诱导肝纤维化小鼠模型中肝脏巨噬细胞及淋巴细胞亚群变化的初步分析

Preliminary analysis of changes of hepatic macrophages and lymphocyte subsets in CCl_4-induced hepatic fibrosis in mice

目的研究CCl_4诱导肝纤维化小鼠模型中肝脏免疫细胞包括巨噬细胞、淋巴细胞亚群的变化。方法 C57BL/6小鼠给予CCl_4腹腔注射建立肝纤维化模型,通过HE和Masson染色评价小鼠肝损伤及纤维化程度。采集小鼠外周血清,检测肝功能指标和血脂水平。取小鼠肝脏制备单个核细胞悬液,用多色流式细胞术检测肝脏免疫细胞亚群的分布。结果实验组小鼠ALT、AST明显高于对照组,TG、LDL-C显著性升高,平均病理评分达到F3级。实验组小鼠库普弗细胞比例(13.33±1.91)%显著高于对照组(8.17±2.00)%,其中以CD11c+标记M1型库普弗细胞比例明显升高,而以CD206+标记M2型库普弗细胞无显著变化。实验组小鼠淋巴细胞中T细胞比例(20.17±4.07)%显著低于对照组(36.00±4.61)%;NKT细胞比例(8.71±1.37)%较对照组(23.41±3.38)%明显降低;而两组间NK细胞和B细胞比例均无明显变化。结论 CCl_4诱导小鼠肝纤维化模型中,肝脏内库普弗细胞的明显增加是以具有促炎效应的M1型巨噬细胞为主,而NKT细胞和T细胞比例则明显减少。M1型巨噬细胞对肝纤维化发展的作用需进一步深入探讨。

Objective To study the changes in the distribution of intrahepatic macrophages and lymphocytes in the mouse model of hepatic fibrosis induced by carbon tetrachloride (CCl4).Methods The hepatic fibrosis model was established in C57BL/6 mice by intraperitoneal injection of CCl4.The degree of hepatic fibrosis was evaluated by hematine-eosin and Masson staining.The peripheral serum of mice was collected to detect liver function index and blood lipid level.The distribution of hepatic immune cell subsets in liver mononuclear cells were analyzed using multi-color flow cytometry.Results Levels of serum alanine transaminase,aspartate aminotransferase,triglyceride and high density lipoprotein cholesterol were significantly higher in CCl4 group than those in control group.CCl4 group had advanced fibrosis (stage 3-4) according to Metavir score.The percentage of Kupffer cells in CCl4 group (13.33±1.91%) was significantly higher than that in control group (8.17±2.00%),in which the percentage of M1 phenotype Kupffer cells with CD11c+ marker increased significantly while the percentage of M2 phenotype Kupffer cells with CD206+ marker had no significant difference.The proportions of T cells and natural killer T (NKT) cells in CCl4 group were significantly lower than those in control group (20.17±4.07%vs.36.00±4.61%,8.71±1.37%vs.23.41±3.38%).While there was no significant difference in the proportions of NK cells and B cells between the 2 groups.Conclusion In CCl4-induced hepatic fibrosis model,the increase of Kupffer cells in liver was mainly caused by M1 macrophages with pro-inflammatory effect,while the proportions of NKT cells and T cells were significantly decreased.The role of M1 macrophages in the development of liver fibrosis needs to be further explored.