上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2017, Vol. 37 ›› Issue (7): 906-.doi: 10.3969/j.issn.1674-8115.2017.07.005

• 论著（基础研究） • 上一篇    下一篇

新型酒精性肝病小鼠模型的建立

黄东东，沃璐璐，阮昕，许雅芊，龚一鸣，杨琳希，李雪川，康月苧，贺明   

  1. 上海交通大学 基础医学院病理生理学教研室，细胞分化与凋亡教育部重点实验室，上海 200025
  • 出版日期:2017-07-28 发布日期:2017-08-25
  • 通讯作者: ?贺明，电子信箱：heming@shsmu.edu.cn
  • 作者简介:?黄东东（1991—），男，硕士生；电子信箱：huangdongdong@sjtu.edu.cn
  • 基金资助:
    国家自然科学基金（81470841）；上海市浦江人才计划（16PJ1405400）

Construction of a new alcoholic liver disease mouse model

HUANG Dong-dong, WO Lu-lu, RUAN Xin, XU Ya-qian, GONG Yi-ming, YANG Lin-xi, LI Xue-chuan, KANG Yue-ning, HE Ming   

  1. Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Basic Medicine Faculty of Shanghai Jiao Tong University, Shanghai 200025, China
  • Online:2017-07-28 Published:2017-08-25
  • Supported by:
    National Natural Science Foundation of China, 81470841; Shanghai Pujiang Program, China, 16PJ1405400

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摘要： 目的 · 建立一种可靠的、模拟酒精性肝病（ALD）患者饮酒模式的新型 ALD 小鼠模型（ALDNM）。 方法 · 在 Lieber-DeCarli 模型基础上，结合Gao-Binge 模型，使用自主设计的饲喂瓶和饲料配方，通过慢性喂养加急性灌胃法建立ALDNM 模型：给予小鼠 5 d 液体对照饲料适应后，随机分为对照组和乙醇组，每组10 只；乙醇组小鼠自第6 日饲喂乙醇占热量比为30% 的饲料10 d，第 16 日上午2 组小鼠各自以31.5% 乙醇或等热量的糊精溶液灌胃，9 h 后收集标本。比较Lieber-DeCarli 模型和ALDNM 模型小鼠的 一般情况、肝脏病理学改变和血清学指标；利用油红O 染色和三酰甘油（TAG）检测，明确ALDNM 小鼠肝脏脂质含量；利用realtime PCR 检测ALDNM 模型肝组织中白介素-6（IL-6）、肿瘤坏死因子α（TNF-α）、脂肪酸合成酶（Fas）、超长链脂肪酸延伸酶6 （Elovl6）、硬脂酰辅酶A 去饱和酶（Scd1）等基因mRNA 水平，Western blotting 检测肝组织蛋白中磷酸化信号转导与转录激活因 子（p-STAT3）的变化。结果 · Lieber-DeCarli 模型小鼠一般状态较差，血清谷丙转氨酶（GPT）、谷草转氨酶（GOT）无明显变化。 ALDNM 模型中，乙醇组小鼠肝细胞极度肿胀呈圆形，体积增大，细胞质内充满大量脂肪空泡；油红 O 染色结果显示，乙醇组小鼠肝 细胞内呈现深浅不等的油红 O 着色；乙醇组小鼠肝重指数、肝脏 TAG 含量、血清 GPT 和 GOT 较对照组显著升高，血清 HDL 明显降 低；而且，ALDNM 乙醇组小鼠肝脏中炎症通路和脂质合成代谢通路相关基因表达水平显著升高。结论 · 成功建立了ALDNM 模型； 该模型操作简单，构建时间短，费用低，能较好地模拟 ALD 患者饮酒模式和发病过程，且具有小鼠进食量平稳、重复性好、肝脏损 伤明显等优点。

关键词: 酒精性肝病, 小鼠, 动物模型, 炎症信号通路, 脂质合成信号通路

Abstract:

Objective · To establish a reliable alcoholic liver disease mouse model (ALDNM) that mimics the drinking pattern of alcoholic liver disease (ALD) patients.  Methods · Using the self-designed feeding tubes and liquid diet, ALDNM model was developed through chronic feeding combined with acute gavage of ethanol based on Lieber-DeCarli model and Gao-Binge model. C57BL/6 mice were administered with control liquid diet for adaptation for first 5 d, and then divided into pair-fed group and ethanol-fed group (10 mice each group). Ethanol-fed mice were fed with the liquid diet in which ethanol accounts for 30% of total energy, while the pair-fed mice were fed with the control diet for 10 d. At the 16th day, ethanol-fed mice and pair-fed mice were respectively gavaged a single dose of 31.5% ethanol or isocaloric maltose dextrin, and euthanized 9 h later. Sera and livers were collected. The general physiological condition, hepatic tissue pathological changes and serum indexes between Lieber-DeCarli models and ALDNM models were compared. The liver lipids of ALDNM mice were determined by Oil red O (ORO) staining and hepatic triacylglyceride (TAG) test. Meanwhile, the mRNA levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), fatty acid synthase (Fas), long chain fatty acid elongase 6 (Elovl6) and stearyl-CoA desaturase (Scd1) were detected by real-time PCR in ALDNM models. Western blotting was used to detect the changes of phosphorylated signal transduction and transcriptional activator (p-STAT3) in the livers.  Results · Lieber-DeCarli model mice were generally in poor condition, and there was no significant change in serum glutamic-pyruvic transaminase (GPT) and glutamic-oxaloacetic transaminase (GOT) compared to pair-fed group. However, in ALDNM models, H-E staining showed that the hepatocytes of ethanol-fed mice were extremely swollen with round volume, increased cytoplasm and filled with large amounts of fat vacuoles. ORO staining analyses showed obvious microsteatosis in the liver cells from all ethanol-fed mice. The hepatosomatic index, liver TAG content, serum GPT and GOT of ALDNM models were significantly higher than those in the pair-fed group, while the serum HDL significantly decreased compared to the pair-fed group. Moreover, the expression levels of both lipid synthesis pathways and inflammatory signaling pathways related genes in livers significantly increased in the ethanol-fed mice of ALDNM model.  Conclusion · ALDNM model was successfully constructed. This model is cost- and time-efficient. Moreover, ALDNM model mimics the drinking pattern and pathogenesis of ALD patients with the advantages of stable food intake, good repeatability, and obvious liver damage.

Key words:  alcoholic liver disease, mouse, animal model, inflammatory signaling pathway, lipid synthesis pathway