非酒精性脂肪性肝炎相关肝细胞癌小鼠模型的建立与鉴定

Establishment and evaluation of a mouse model of nonalcoholic steatohepatitis-related hepatocellular carcmoma

目的用高脂高胆固醇饮食喂养载脂蛋白E和低密度脂蛋白受体双基因敲除（ApoE-／-/LDLR-/-）小鼠，构建-种基于基因敲除小鼠的非酒精性脂肪性肝炎相关肝癌模型。方法首先用两种基因敲除小鼠ApoE√和LDLR-’交配，从后代中鉴定筛选出ApoE-／-/LDLR-/-纯合子小鼠。纯合子小鼠交配繁殖的子代在出生后2～3d内注射链脲佐菌素（STZ），断乳后继续高脂高胆固醇饮食至第20周。同时设立对照组ApoE-／-/LDLR-/-小鼠断乳后普通饮食。并于第10、16、20周末分批处死小鼠，检测体质量及生物化学指标。肝组织HE、油红O染色和Masson染色评价病理学改变。免疫组织化学分析肝癌标志物磷脂酰肌醇蛋白聚糖3（Glypican3）的表达情况。结果模型组小鼠空腹血糖、总胆固醇水平均明显高于对照组（P〈0．01），丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和总甘油三酯随造模时间延长逐渐增加，在20周时差异均有统计学意义（P〈0．05）。与对照组相比较，HE染色结果显示模型组小鼠10周出现酒精性脂肪性肝炎，16周出现异型增生结节，20周出现早期肝癌。肝脏油红0染色面积、密度、染色强度等结果提示在10周酒精性脂肪性肝炎阶段脂肪变程度最高，而后随造模时间延长脂肪变程度相对减轻。Masson染色结果显示模型组小鼠16周有轻度窦周纤维化，20周癌组织周围纤维化，有纤维间隔形成。对照组HE染色、油红O染色、Masson染色均未见异常。免疫组织化学结果显示模型组小鼠16周Glypican3在结节部位表达阳性，20周在肝癌组织细胞质表达阳性。结论新出生ApoE-／-/LDLR-/-小鼠经STZ注射后高脂高胆固醇饮食20周可以模拟脂肪肝相关肝细胞癌的疾病进程，可能不经过肝硬化直接发展成为早期肝癌。

Objective To establish an apolipoprotein E （ApoE） and low-density lipoprotein receptor （LDLR） double-knockout （ApoE//LDLR/） mouse model of nonalcoholic steatohepatitis （NASH）-related hepatoceUular carcinoma （HCC） induced by high-fat and high-cholesterol （HFHC） diet. Methods ApoE-/knockout mice were crossed with LDLR-/- knockout mice to obtain ApoE-/-/LDLR-/- mice. The ApoE-/-/LDLR-/- mice mated with each other, and the offspring were injected with low-dose streptozotocin （STZ） at 2-3 days aider birth. Some mice were fed with HFHC diet after weaning as the model group （n = 15）, and some mice were fed with normal diet as the control group （11 = 15）. Mice were sacrificed at the end of weeks 10, 16, and 20 （5 mice at each time point）. The body weight was measured. Liver tissue and blood were collected to measure biochemical parameters, evaluate the pathological changes in the liver tissue by HE staining, oil red O staining, and Masson staining, and detect the expression of glypican-3 （a marker of HCC） by immunohistochemical staining. Results The model group had significantly higher levels of fasting blood glucose and total cholesterol than the control group （P 〈 0.01）. Serum levels of alanine aminotransferase, aspartate aminotransferase, and total triglycefide gradually increased with time in the model group; at week 20, there were significant differences in above three indices between the two groups （P 〈 0.05）. HE staining showed that compared with the control group at the corresponding time point, the model group developed sequential histological changes： NASH at week 10, dysplastic nodules at week 16, and early HCC at week 20. Oil red O staining showed that in the model group, the degree of liver steatosis increased within 10 weeks and gradually decreased later. Masson staining demonstrated that the model group developed pathological changes： mild perisinusoidal fibrosis at week 16 and bridging fibrosis around tumors at week 20. HE staining, oil red O staining, and Masson staining showed that no histological or pathological changes were found in the control group. Glypican-3 was detected in the nodules at week 16 and in the cytoplasm of HCC cells at week 20 in the model group. Conclusion The mouse model of NASH-related HCC can be developed by giving STZ injection to neonatal ApoE-/-/LDLR-/- mice and feeding them with HFHC diet after weaning for 20 weeks. Early HCC may develop directly from NASH.