Research for establishing a mouse model of cholangiocarcinoma with bile duct ligation of left and middle lobe of liver and diethylnitrosamine

Objective To prepare for a mouse model of human cholangiocarcinoma usingdiethylnitrosamine (DEN) combined with left and middle bile duct ligation. Methods One hundred and twenty male Balb/c mice aged 6-7 weeks were studied to make an animal model, and divided into five groups: group DLD (n=30, DEN+biliary ligation), group BLANK (n=20, blank control), group DEN (n=20, only DEN treatment), group LMBDL (n=30, bile duct ligation), group DSO (n=20, DEN+cholecystectomy). The body weight, liver appearance, survival rate and tumor formation rate of each group were recorded and compared at 0, 2, 4, 8, 12, 16, 20, 24 weeks after modeling. Hematoxylin-eosin (HE) staining and immunohistochemistry were used to detect the expression of biliary epithelial markers AQP-1 and CK19 in the liver tissues of each tumor nodule or non-tumor mice to identify the cells source of the tumor nodules. Results (1) There was no significant difference in body weight between group BLANK and group LMBDL (t=0.572, P=0.611). The body weight of group DEN was significantly lower than that of group BLANK (t=3.382, P=0.002), group DLD lower than that of group DEN and group DSO (t=2.022, 2.331; P=0.019, 0.021), which suggested that bile duct ligation had no significant effect on the body weight of mice, while DEN administration and combined partial bile duct ligation both had significant effects on body weight. (2) Compared with the survival rate of group BLANK, groups DEN, DLD, DSO and LMBDL showed no statistically differences (P=0.058, 0.013, 0.025, and 0.692, respectively), indicating that bile duct ligation and DEN did not increase mortality in mice. (3) Mice in groups BLANK, DEN, and LMBDL did not form tumors, and tumor formation rates of groups DSO and DLD were 8.3% (1/12) and 47.1% (9/17), respectively. The tumor formation rate of group DLD was higher than the other four groups (all P<0.01). There was no distant metastasis in each group, suggesting that bile duct ligation combined with DEN can increase the tumor formation rate in mice. (4) CK19 and AQP-1 were strongly positive in the tumor nodules of group DLD, which indicated the source of biliary cells. Conclusions Low dose of DEN with left and middle bile duct ligation can induce the formation of cholangiocarcinoma. The model has the characteristics of simple operation, high tumorigenesis rate and survival rate, and conforms to the occurrence and development process of human cholangiocarcinoma. Key words: Cholestasis; Diethylnitrosamine; Bile duct neoplasms; Models, animal