运用二乙基亚硝胺（DEN）联合肝左、中叶胆管结扎方法，制作可供研究、重复性高、模拟人胆管癌发生过程的小鼠模型。

将120只6~7周龄Balb/c雄性小鼠制作成动物实验模型：DLD组（n=30，DEN+胆管结扎）、BLANK组（n=20，空白对照）、DEN组（n=20，仅DEN处理）、LMBDL组（n=30，胆管结扎）、DSO组（n=20，DEN+假手术）。分别于造模后0、2、4、8、12、16、20、24周时，记录和比较各组小鼠体质量、肝脏外观差异、存活率、成瘤率；采用苏木精-伊红（HE）染色和免疫组织化学法检测各组成瘤结节或未成瘤小鼠肝脏组织中胆管上皮标志物AQP-1、CK19的表达，明确成瘤结节的细胞来源。

（1）BLANK组与LMBDL组小鼠的体质量差异无统计学意义（t=0.572，P=0.611），DEN组体质量显著低于BLANK组（t=3.382，P=0.002），DLD组体质量均低于DEN组及DSO组（t=2.022、2.331，P=0.019、0.021），单纯胆管结扎对小鼠体质量变化无显著影响，而DEN给药以及联合部分胆管结扎均对小鼠体质量产生影响。（2）DEN、DLD、DSO、LMBDL组的存活率与BLANK组相比，差异无统计学意义（P=0.058、0.013、0.025、0.692），胆管结扎手术与DEN不会增加小鼠死亡率。（3）BLANK、DEN、LMBDL组小鼠均未成瘤，DSO、DLD组的成瘤率分别为8.3%（1/12）和47.1%（9/17），DLD组的成瘤率高于其他四组（均P<0.01），各组均未见远处转移瘤，胆管结扎联合DEN可提高小鼠成瘤率。（4）CK19及AQP-1在DLD组小鼠成瘤结节中表达均为强阳性，结节为胆管细胞来源。

低剂量DEN联合肝左、中叶胆管结扎可成功构建胆管癌模型，该模型具有操作简单、成瘤率及存活率高、符合人胆管癌发生发展过程等特点。

To prepare for a mouse model of human cholangiocarcinoma usingdiethylnitrosamine (DEN) combined with left and middle bile duct ligation.

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.

(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.

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.