ACM Advances in Clinical Medicine 2161-8712 Scientific Research Publishing 10.12677/ACM.2022.12121708 ACM-59678 ACM20221200000_85232593.pdf 医药卫生 乙醇喂养小鼠诱导肝损伤模型的制作 Establishment of Liver Injury Model in Mice Fed with Ethanol 黄 世豪 2 1 林 丽珍 3 1 李 永龙 4 2 1 夏 加伟 5 1 赵 文淘 6 1 韩 留鑫 5 1 李 迎春 4 2 1 戴 冠齐 2 1 何 丹华 2 1 丛 金格 4 2 1 沈 含章 5 1 贾 俊双 2 1 林 晓琳 2 1 肖 东 4 2 1 申 红芬 2 1 南方医科大学基础医学院肿瘤研究所，广东 广州；南方医科大学实验动物管理中心，广东 广州 南方医科大学基础医学院肿瘤研究所，广东 广州 南方医科大学检验与生物技术学院，广东 广州 昆明医科大学第三附属医院(云南省肿瘤医院，云南省癌症中心)消化肿瘤内科，云南 昆明 昆明市第三人民医院(大理大学第六附属医院)，云南 昆明 null 05 12 2022 12 12 11858 11866 © Copyright 2014 by authors and Scientific Research Publishing Inc. 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

目的：通过慢性酒精喂养加急性酒精灌胃来制作肝损伤的小鼠模型。方法：将雄性C57BL/6小鼠随机分为对照组和模型组。前十天每天给予对照组不含酒精液体饲料喂养，给予模型组酒精液体饲料喂养，第十一天给对照组灌胃麦芽糊精溶液(20 μL/Kg)，模型组灌胃酒精(20 μL/Kg)。实验结束后检测血清肝功能指标、脂质代谢相关指标，并观察肝脏组织病理变化。结果：与对照组小鼠比较，模型组小鼠血清中胆固醇(P < 0.0001，n = 14~15)、甘油三酯(P < 0.01, n = 14~15)和谷丙转氨酶(P < 0.01, n = 14~15)均显著升高；肝脏匀浆检测胆固醇和甘油三酯(P > 0.05, n = 14~15)在实验组中均高于对照组，且胆固醇(P < 0.01, n = 14~15)在两组间有显著差异。苏木精–伊红结果显示，与对照组相比，造模组的肝组织破坏程度较重。结论：模型组小鼠出现了肝细胞损伤，脂肪累积，运用慢性喂养加上急性灌胃的方法成功模拟酒精肝损伤，这对于之后的临床治疗和新机制的研究具有实践意义。 Objective: A mouse model of liver injury was created by chronic alcohol feeding and acute alcohol gavage. Method: Male C57BL/6 mice were randomly divided into control group and model group. For the first ten days, the control group was fed with control liquid feed, and the experimental group was fed with alcohol liquid feed. On the 11th day, the control group was fed with maltodextrin solution (20 μL/kg), and the model group was fed with alcohol (20 μL/kg). After the experiment, serum liver function indexes and lipid metabolism-related indexes were detected, and the patho-logical changes of liver tissue were observed. Result: Compared with the control group, the serum cholesterol (P < 0.0001, n = 14~15), triglyceride (P < 0.01, n = 14~15) and alanine aminotransfer-ase (P < 0.01, n = 14~15) of the model group were significantly higher than those of the control group. Significantly increased; cholesterol and triglyceride (P > 0.05, n = 14~15) detected in liver homogenate were higher in the experimental group than in the control group, and there was a sig-nificant difference in cholesterol (P < 0.01, n = 14~15) between the two groups. The results of he-matoxylin-eosin showed that compared with the control group, the damage degree of liver tissue in the model group was more serious. Conclusion: The mice in the model group had liver cell damage and fat accumulation. The method of chronic feeding and acute gavage was used to successfully simulate alcoholic liver injury, which has practical significance for the subsequent clinical treat-ment and the study of new mechanisms.

酒精性肝损伤，苏木精伊红染色，小鼠模型, Alcohol Liver Disease Hematoxylin Eosin Staining Mice Model 摘要

目的：通过慢性酒精喂养加急性酒精灌胃来制作肝损伤的小鼠模型。方法：将雄性C57BL/6小鼠随机分为对照组和模型组。前十天每天给予对照组不含酒精液体饲料喂养，给予模型组酒精液体饲料喂养，第十一天给对照组灌胃麦芽糊精溶液(20 μL/Kg)，模型组灌胃酒精(20 μL/Kg)。实验结束后检测血清肝功能指标、脂质代谢相关指标，并观察肝脏组织病理变化。结果：与对照组小鼠比较，模型组小鼠血清中胆固醇(P < 0.0001，n = 14~15)、甘油三酯(P < 0.01, n = 14~15)和谷丙转氨酶(P < 0.01, n = 14~15)均显著升高；肝脏匀浆检测胆固醇和甘油三酯(P > 0.05, n = 14~15)在实验组中均高于对照组，且胆固醇(P < 0.01, n = 14~15)在两组间有显著差异。苏木精–伊红结果显示，与对照组相比，造模组的肝组织破坏程度较重。结论：模型组小鼠出现了肝细胞损伤，脂肪累积，运用慢性喂养加上急性灌胃的方法成功模拟酒精肝损伤，这对于之后的临床治疗和新机制的研究具有实践意义。

 关键词

酒精性肝损伤，苏木精伊红染色，小鼠模型

 Establishment of Liver Injury Model in Mice Fed with Ethanol<sup> </sup>

Shihao Huang1*, Lizhen Lin2*, Yonglong Li1,3, Jiawei Xia4, Wentao Zhao5, Liuxin Han4, Yingchun Li1, Guanqi Dai1, Danhua He1, Jing’e Cong1,3, Hanzhang Shen4, Junshuang Jia1, Xiaolin Lin1, Dong Xiao1,3, Hongfen Shen1#

1Cancer Research Institute, School of Basic Medical Science, Southern Medical University, Guangzhou Guangdong

2School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou Guangdong

3Institute of Comparative Medicine and Laboratory Animal Center, Southern Medical University, Guangzhou Guangdong

4The Third People’s Hospital of Kunming (The Sixth Affiliated Hospital of Dali University), Kunming Yunnan

5Department of Gastrointestinal Oncology, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Yunnan Cancer Center), Kunming Yunnan

Received: Nov. 23rd, 2022; accepted: Dec. 21st, 2022; published: Dec. 28th, 2022

 ABSTRACT

Objective: A mouse model of liver injury was created by chronic alcohol feeding and acute alcohol gavage. Method: Male C57BL/6 mice were randomly divided into control group and model group. For the first ten days, the control group was fed with control liquid feed, and the experimental group was fed with alcohol liquid feed. On the 11th day, the control group was fed with maltodextrin solution (20 μL/kg), and the model group was fed with alcohol (20 μL/kg). After the experiment, serum liver function indexes and lipid metabolism-related indexes were detected, and the pathological changes of liver tissue were observed. Result: Compared with the control group, the serum cholesterol (P < 0.0001, n = 14~15), triglyceride (P < 0.01, n = 14~15) and alanine aminotransferase (P < 0.01, n = 14~15) of the model group were significantly higher than those of the control group. Significantly increased; cholesterol and triglyceride (P > 0.05, n = 14~15) detected in liver homogenate were higher in the experimental group than in the control group, and there was a significant difference in cholesterol (P < 0.01, n = 14~15) between the two groups. The results of hematoxylin-eosin showed that compared with the control group, the damage degree of liver tissue in the model group was more serious. Conclusion: The mice in the model group had liver cell damage and fat accumulation. The method of chronic feeding and acute gavage was used to successfully simulate alcoholic liver injury, which has practical significance for the subsequent clinical treatment and the study of new mechanisms.

Keywords:Alcohol Liver Disease, Hematoxylin Eosin Staining, Mice Model

Copyright © 2022 by author(s) and beplay安卓登录

This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).

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 1. 前言

酒精性肝病(Alcohol liver disease, ALD)是长期大量饮酒引起的肝脏损伤，肝组织会出现肝纤维化(Fibrosis)，肝硬化(Cirrhosis)、肝细胞癌(Hepatocellular carcinoma, HCC)的病理变化 [ 1 ]。这些特征不是疾病的不同阶段，可以在同一人身上共存 [ 2 ]。到2013年，中国已成为仅次于英国的全球第二大饮酒大国。ALD患病率由2000年的2.27%上升到2015年的8.74%，增加了3.8倍 [ 3 ]。

为了更好地了解ALD，目前有三种常用的肝损伤动物模型，包括使用最广泛的慢性自由摄入乙醇模型(Lieber-DeCarli液体饮食模型)、慢性灌胃乙醇(Tsukamoto-French模型)，以及慢性加暴食乙醇(高斌国家酒精滥用和酒精中毒研究所(NIAAA)模型) [ 4 ]。1963年Lieber和Decarli配制出含酒精和全营养素的液体食料，首创Lieber-DeCarli模型 [ 5 ]。造模动物只喂食该液体饲料，持续4~6周。然而，在没有继发性损伤的情况下，该模型仅引起轻度脂肪变性，血清丙氨酸转氨酶轻微升高，很少或没有炎症反应。慢性灌胃乙醇(Tsukamoto-French模型)通过持续灌胃诱导严重脂肪变性、轻度肝炎症和轻度纤维化 [ 6 ]。虽然该模型对ALD发病机制的研究非常有用，但由于其技术难度大，且需要重症监护和昂贵的设备，其应用范围有限。与上述的啮齿类动物模型相比，NIAAA模型 [ 7 ] 可以设置在大多数实验室和在较短的时间内进行，拥有时间成本和效率。这种模型，使用慢性饲养加一次急性灌胃的方法，能显著提高小鼠血清ALT和AST水平，这也密切再现了ALD患者的饮酒行为和在患者身上的急性–慢性肝损伤。

C57BL/6小鼠是自由摄取酒精的最佳品系 [ 7 ]，因为其他品系可能需要更长时间的驯化才能确保足够的摄入量，或可能在实验完成前由于酒精摄入而有较高的死亡率。

因此本实验用C57小鼠，先适应液体饲料5天，再慢性喂养含有5%酒精的液体饲料或不含酒精的饲料10天，第十一天同浓度的灌胃乙醇或麦芽糖溶液，以完成造模，灌胃9小时后取材如图1，获得血清和肝脏，将肝脏研磨成匀浆，将材料送去检测脂质代谢的指标胆固醇、甘油三酯和肝损伤的指标ALT和AST，并对组织切片进行HE染色，并在显微镜下观察结果。期望制作成功酒精性肝损伤的动物模型，以用于后续ALD相关研究。

图1. 酒精肝损伤小鼠模型构建示意图

 2. 材料与方法 2.1. 材料与试剂 2.1.1. 小鼠

本实验所用小鼠品系为C57BL/6 (下文简称C57小鼠)，43~49日龄，SPF级，雄性，购自南方医科大学实验动物管理中心。所有实验用小鼠均在SPF级环境中饲养和繁殖，温度控制在22℃，12小时昼夜循环的合理生物节律，自动光控，自由摄食。

 2.1.2. 主要试剂

1%戊巴比妥钠、无水乙醇、麦芽糖糊精磷酸盐缓冲液(PBS)二甲苯、无水乙醇、95%乙醇、90%乙醇、80%乙醇、70%乙醇、50%乙醇、双蒸水(dd水)、1%盐酸乙醇、自来水、苏木素染液、伊红染液、中性树胶、4%多聚甲醛、石蜡。

 2.2. 方法 2.2.1. 动物分组

小鼠分10笼，每笼3只，随机分组并标号，24 h昼夜周期喂养。待小鼠9周龄时，开始适应性喂养不含酒精液体饲料5 d。正式开始分组喂养含酒精饲料和不含酒精饲料之前，根据当天体重，随机将小鼠分为实验组和对照组。

 2.2.2. NIAAA模型制备及一般体征检测

第1~10天对照组小鼠给予含酒精液体饲料，实验组小鼠给予标准Lieber-Decarli饲料。第11天，禁食6小时后，对照组小鼠灌胃麦芽糖糊精溶液，实验组小鼠灌胃酒精。第一天给予50 mL饲料，后续每天给两组等量(实验组前一天消耗的饲料量)的饲料。每天定时测定小鼠的体重及进食量。

 2.2.3. 小鼠取材及处理

用1%戊巴比妥钠麻醉小鼠后取血，将小鼠处死，取肝组织研磨，于入−80℃冰箱待用。

 2.2.4. 石蜡切片HE染色

收取离体后的小鼠肝脏，置于4%的福尔马林固定，梯度酒精脱水，二甲苯透明，浸蜡，常规石蜡包埋，3~4 μm连续切片，组织切片进行HE染色。

 2.2.5. 脂肪变程度判断

借鉴NAS积分(NAFLD activity score, NAS)中的方法，根据脂肪变细胞所占肝细胞的百分比来评估脂肪变性程度。脂肪变细胞占肝细胞的总数小于5%，判定为S0；占5%~33%，判定为S1；占34%~66%，判定为S2；多于67%，判定为S3。

 2.2.6. 统计学分析

本实验数据用SPSS 17.0进行统计学分析，两组间比较用配对样本T检验，采用卡方检验研究不同组别、肝损伤轻微和肝损伤程度严重是否存在差异，用Graphpad Prism 8.0绘图。P < 0.05为有统计学差异。

 3. 结果 3.1. 小鼠肝指数的变化

根据1~11天小鼠的进食量及体重统计两组小鼠体重变化(图2(a))，两组小鼠肝组织的相对重量(图2(b))。在造模的过程中，小鼠每日进食量处于波动状态，总体来说还是对照组比实验组动物摄入多一些，对照组小鼠的体重大多高于实验组小鼠。而实验组小鼠肝重体重比的数值明显大于对照组，两组间有显著差异(P < 0.001, n = 14~15)。

 3.2. 血清中ALT和AST检测结果

处理后的血清检测谷丙转氨酶、谷草转氨酶。与对照组相比，谷丙转氨酶(P < 0.01, n = 14~15)在对照组小鼠中也是显著有升高的，但在两组小鼠中谷草转氨酶(P > 0.05, n = 14~15)无明显差异(图3)。

 3.3. 血清及肝脏中总胆固醇和甘油三酯检测结果

处理后的肝脏匀浆及血清检测总胆固醇和甘油三酯。与对照组相比，实验组小鼠的血清中，甘油三酯(P < 0.0001, n = 14~15)和总胆固醇(P < 0.01, n = 14~15)显著升高；实验组小鼠肝脏中总胆固醇和甘油三酯(P > 0.05, n = 14~15)均升高，而其中总胆固醇(P < 0.001, n = 14~15)在两组中有显著差异(图4)。

图2. 模型组的肝指数增加

图3. 血清中ALT和AST含量变化

图4. 血清和肝组织中胆固醇与甘油三脂含量变化

 3.4. 肝组织切片HE染色

正置显微镜20倍物镜下，对照组细胞核大而圆，居中，异染色质少而着色浅，能清晰看到核仁，胞浆嗜酸性，呈红色。造模组小鼠出现了肝细胞坏死、混合性脂肪变、水肿等现象(图5)。

 3.5. 脂肪变程度统计

根据NAS评分，给对照组和造模组小鼠的HE染色切片分级，由低到高，从S0~S3。经SPSS卡方检验后可以得知对照组和造模组之间的脂肪变严重程度之间是有显著差异(P < 0.01, n = 14~15)。在14个对照组小鼠中，有12个(85.7%)被评估为脂肪变程度低，2个(14.2%)被判定为脂肪变程度高。与之相比，在15个造模组小鼠中，5个(33.3%)被判定为脂肪变性不严重，10个(66.7%)被认为脂肪变性程度较为严重，见表1。

图5. 肝组织HE染色变化

The result of chi-square test of fat change degre
组别 脂肪变程度(%) 总计 c2 P
S0~S1 S2~S3
Con mouse 12 (85.7%) 2 (14.2%) 14 8.191 0.008
NIAAA mouse 5 (33.3%) 10 (66.7%) 15
总计 17 12 29

表1. 脂肪变程度卡方检验结果

 4. 讨论分析

酒精性肝损伤是由于长期大量饮酒引起的肝脏损伤。酒精性肝硬化占肝硬化的病因构成比从1999年的10.8%上升到2003年的24.0%。酒精性肝病已成为我国最主要的慢性肝病之一 [ 8 ]。为了深入研究酒精性肝损伤，研究疾病的发病机制，信号通路等，研究人员开始尝试制作ALD的动物疾病模型 [ 9 ] - [ 14 ]。综上所述，NIAAA是比较理想的方法。因此本实验采用了这个方法进行肝损伤小鼠模型的制作。

肝炎症升高和肝损伤是临床ALD患者的标志 [ 15 ]。为了验证模型制作的结果，除了检测组织、血清中特定指标，我们用病理组织切片染色证明肝脏组织出现异常情况。造模组中出现大泡性脂肪变、小泡性脂肪变、水肿等现象。

小鼠进食量处于波动状态，对照组比造模组动物摄入多，体重大多高于造模组小鼠，造模组小鼠的肝重/体重比明显大于对照组，可能是由于造模组饲料含有酒精，导致小鼠食欲下降，体重下降，酒精摄入对肝细胞有毒害作用，导致肝功能受损。

与对照组相比，造模组的血清和肝组织中甘油三酯和总胆固醇显著升高，提示肝脏中有脂肪积累。可能是摄入酒精损害了肝细胞的功能，影响脂肪酸代谢导致脂肪累积。造模组ALT显著升高，AST两组小鼠无明显差异可能是由于只有肝细胞胞浆受到了损伤，线粒体还未损伤，或损伤不明显 [ 16 ]。上述结果表明造模组小鼠出现酒精性肝损伤。

通过NIAAA的方法，我们成功造模出酒精性肝损伤的动物模型。建立与临床肝损伤的病理生理过程及发病程度相似的动物模型对于更好地探究肝损伤患者的发病机制及临床治疗方法具有十分重要的实践意义、现实意义。通过动物模型实验，我们可以深入地研究我们探究的基因与肝损伤之间的关联。且这种能够模拟人类饮酒模式和酒精诱导的肝脏病理的范围和严重程度的动物模型在我们寻找肝损伤新机制和治疗靶点的过程中也至关重要。

 基金项目

广东省基础与应用基础研究基金(2022A1515012477)，METTL14通过m6A甲基化修饰脂肪酸转位酶FAT调控脂肪肝形成的研究。

广东省医学科研基金(B2014238)，小鼠诱导性多潜能干细胞(mIPScell)对肝损伤修复的研究。

1) 国家自然科学基金(82060425)，miR-26a-5p靶向转录因子DCUN1D1调控个旧矿工肺癌进展的研究。

2) 云南省基础研究专项基金(202201AT070044)，ZBTB48靶向MTP18促进线粒体分裂抑制肝癌细胞存活和干性的机制研究。

3) 云南省科技厅地方高校联合专项基金(202001BA070001-043)，蛋白激酶MST4负向调控PI3K/AKT通路抑制肺癌迁移和侵袭的机制研究。

4) 云南省科技厅地方高校联合专项基金(202001BA070001-063)，利用肝脏特异性敲除ZNF173的转基因小鼠解析转录因子ZNF173在肝癌发生中的作用。

 文章引用

黄世豪,林丽珍,李永龙,夏加伟,赵文淘,韩留鑫,李迎春,戴冠齐,何丹华,丛金格,沈含章,贾俊双,林晓琳,肖 东,申红芬. 乙醇喂养小鼠诱导肝损伤模型的制作Establishment of Liver Injury Model in Mice Fed with Ethanol[J]. 临床医学进展, 2022, 12(12): 11858-11866. https://doi.org/10.12677/ACM.2022.12121708

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