非酒精性脂肪肝病模型小鼠的肝脏病理变化及肝细胞获取方法

Liver pathological changes and hepatocyte acquisitionmethod of model mice with non-alcoholic fatty liver disease

目的检测非酒精性脂肪肝病(NAFLD)模型小鼠的肝脏病理变化,并探索一种获得大量高活性、高纯度的NAFLD小鼠原代肝细胞的实验方法。方法60只小鼠随机分为正常组(正常饲料,CD)和高脂组(高脂饲料,HFD),分饮食饲养20周。定期检测小鼠的体质量,苏木精-伊红(HE)染色和天狼猩红染色观察肝脏组织病理特点。高脂饮食15周时,原位灌注Ⅳ型胶原酶6~8 mL消化得到混合肝细胞,铺于40%的Percoll分离液上,进行密度梯度离心获得纯化的肝细胞,锥虫蓝染色确定细胞的活性,显微镜观察其形态。结果随着建模时间的延长,HFD组小鼠的体质量逐渐增加,且增幅显著高于CD组;HFD组小鼠肝脏脂肪变性程度增加,在第20周伴随出现了纤维化;建模15周时,每只NAFLD小鼠可获得5×10^(7)~7×10^(7)个肝细胞,细胞存活率≥85%,细胞纯度≥95%,细胞呈圆形或椭圆形,其边缘平滑,且聚团少;通过精准控制温度、降低胶原酶浓度、使用低速离心和低速密度梯度离心等优化条件后,获得的细胞数量和质量均有显著提高。结论成功构建了NAFLD小鼠模型,并揭示了不同疾病阶段肝脏的病理特点。探索了一种高效、稳定的获取NAFLD小鼠原代肝细胞的方法。所得肝细胞的数量和活性都符合大多数实验的要求。

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 collagenaseⅣwere 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 fibrosiscould be detected at week 20.At week 15,about 5×10^(7)-7×10^(7) 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.