关键词: 非酒精性脂肪肝病, 小鼠模型, 病理学

Abstract: Objective To detect the pathological changes in the liver of mice with non-alcoholic fatty liver disease (NAFLD) and explore an experimental method to obtain a large number of primary hepatocytes with high activity and purity from the mice with NAFLD. Methods A total of 60 mice were randomly divided into control group (chow diet, CD) and high-fat group (high-fat diet, HFD) and had been fed separately for 20 weeks. The body weight of the mice was regularly measured and the pathological characteristics of the liver tissue were observed by hematoxylin-eosin (HE) staining and Sirius red staining. After 15 weeks of high-fat diet, 6-8 mL collagenase Ⅳ were added for digestion to obtain mixed hepatocytes, which were spread on 40% Percoll separation solution and subjected to density gradient centrifugation to obtain purified hepatocytes. The viability of the cells was determined by trypan blue staining, and the morphology was observed by microscope. Results With the prolongation of modeling time, the body weight of mice in the HFD group were gradually increased, which was significantly higher than that in the CD group. In the HFD group, the liver of mice gradually developed fatty degeneration, and liver fibrosis could be detected at week 20. At week 15, about 5×10⁷-7×10⁷ hepatocytes could be obtained from each NAFLD mouse with cell viability ≥85% and cell purity ≥95%. Cells were round or elliptical, with smooth edges and few clusters. The number and quality of cells obtained were significantly improved by optimizing conditions such as accurate temperature control, reduced the concentration of collagenase, and the use of low-speed centrifugation and low-speed density gradient centrifugation. Conclusions The NAFLD mouse model was successfully established and the pathological characteristics of liver in different disease stages were revealed. An efficient and stable method for obtaining primary hepatocytes of NAFLD mice was established. The number and activity of hepatocytes obtained met the requirements of most experiments.

Key words: non-alcoholic fatty liver disease, mouse model, pathology