上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2025 , Vol. 45 >Issue 11: 1480 - 1489

DOI: https://doi.org/10.3969/j.issn.1674-8115.2025.11.007

论著 · 基础研究

羧酸酯酶1f基因活体肝转染对急性肝衰竭影响的研究

  • 边姝 ,
  • 于倩 ,
  • 张钰婷 ,
  • 李玎嘉 ,
  • 张乔婷 ,
  • 刘亮明
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  • 1.南京医科大学上海松江临床医学院感染科，上海 201600
    2.南京医科大学上海松江临床医学院消化科，上海 201600
    3.上海交通大学医学院附属松江医院感染科，上海 201600
刘亮明，教授、主任医师，博士；电子信箱：liuliangming@shsmu.edu.cn。

收稿日期: 2025-02-20

  录用日期: 2025-07-03

  网络出版日期: 2025-12-03

基金资助

国家自然科学基金(81770612);国家自然科学基金(81070357);上海市松江区科委科技攻关项目(2023SJKJGG027);上海市松江区公共卫生体系建设三年行动计划(SJGW6-08)

收起

Research on the impact of in vivo liver transfection of carboxylesterase 1f gene on acute liver failure

  • BIAN Shu ,
  • YU Qian ,
  • ZHANG Yuting ,
  • LI Dingjia ,
  • ZHANG Qiaoting ,
  • LIU Liangming
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  • 1.Department of Infection, Shanghai Songjiang Clinical Medical College of Nanjing Medical University, Shanghai 201600, China
    2.Department of Gastroenterology, Shanghai Songjiang Clinical Medical College of Nanjing Medical University, Shanghai 201600, China
    3.Department of Infection, Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 201600, China
LIU Liangming, E-mail: liuliangming@shsmu.edu.cn.

Received date: 2025-02-20

  Accepted date: 2025-07-03

  Online published: 2025-12-03

Supported by

National Natural Science Foundation of China(81770612);Science and Technology Research of Songjiang District Science and Technology Commission in Shanghai(2023SJKJGG027);The Three-Year Action Plan for the Construction of the Public Health System in Songjiang District, Shanghai(SJGW6-08)

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摘要

目的·探究羧酸酯酶1f（carboxylesterase 1f，Ces1f）体内基因转染对脂多糖（lipopolysaccharide，LPS）联合D-氨基半乳糖（D-galactosamine，D-GalN）诱导的急性肝衰竭（acute liver failure，ALF）小鼠的作用，以及对肝脏内源性大麻素（endocannabinoid，EC）及其特异性受体大麻素受体1（cannabinoid receptor 1，Cb1r）和大麻素受体2（cannabinoid receptor 2，Cb2r）表达的影响。方法·将20只C57BL/6J雄性小鼠随机分为4组：空白对照组、Ces1f过表达组、ALF模型组（LPS/D-GalN）和Ces1f过表达ALF模型组（每组n=5）。对于Ces1f过表达模型组通过尾静脉注射含有Ces1f序列的重组腺相关病毒8（recombinant AAV8，rAAV8），对照组注射病毒空载体。病毒注射3周后，采用LPS+D-GalN腹腔注射法构建ALF动物模型。利用免疫荧光观察小鼠肝内Ces1f转染效率，通过实时荧光定量聚合酶链式反应（quantitative real - time PCR，qPCR）和蛋白质印迹法（Western blotting，WB）测定小鼠肝组织CES1F、CB1R和CB2R的表达水平。运用苏木精-伊红（hematoxylin and eosin，HE）染色观察小鼠肝组织病理改变情况。采用酶联免疫吸附法（enzyme - linked immunosorbent assay，ELISA）检测血清丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）、白细胞介素-1β（interleukin-1β，IL-1β）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、花生四酰乙醇酰胺（arachidonoylethanolamide，AEA）和2-花生四酰甘油（2-arachidonoylglycerol，2-AG）水平。结果·AAV8病毒在小鼠肝内转染效率高，具有显著肝趋向性。与对照组相比，ALF模型组Ces1f和Cb2r的mRNA（均P<0.001）及蛋白表达水平（P=0.039，P=0.012）降低，肝组织病理损伤评分，血清ALT、AST、IL-1β和TNF-α水平，肝组织Il-1β mRNA、Tnf-α mRNA、AEA、2-AG、Cb1r mRNA和CB1R蛋白水平显著升高（均P<0.001）。与ALF模型组相比，Ces1f过表达ALF模型组Ces1f mRNA（P<0.001）和CES1F蛋白（P=0.002）表达水平显著升高，血清AST水平降低（P=0.011），肝组织病理损伤评分改善（P=0.001），血清ALT、IL-1β和TNF-α水平，肝组织Il-1β mRNA、Tnf-α mRNA、AEA和2-AG水平显著降低（均P<0.001），Cb1r mRNA（P=0.024）和CB1R蛋白（P=0.027）水平降低。结论·肝靶向性Ces1f基因转染对LPS/D-GalN诱导的ALF小鼠模型具有保护作用，这种保护作用可能是通过下调肝内源性大麻素AEA和2-AG水平和抑制CB1R表达实现。

关键词： 急性肝衰竭; 羧酸酯酶1f; 活体肝转染; 内源性大麻素系统; 小鼠

本文引用格式

边姝 , 于倩 , 张钰婷 , 李玎嘉 , 张乔婷 , 刘亮明 . 羧酸酯酶1f基因活体肝转染对急性肝衰竭影响的研究[J]. 上海交通大学学报（医学版）, 2025 , 45(11) : 1480 -1489 . DOI: 10.3969/j.issn.1674-8115.2025.11.007

Abstract

Objective ·To explore the effects of in vivo gene transfection of Carboxylesterase 1f (Ces1f) on mice with acute liver failure (ALF) induced by lipopolysaccharide (LPS) in combination with D - galactosamine (D - GalN), as well as its impact on the expression of hepatic endocannabinoid (EC) and their specific receptors, cannabinoid receptor 1 (Cb1r) and cannabinoid receptor 2 (Cb2r). Methods ·Twenty male C57BL/6J mice were randomly divided into four groups (n=5 per group): the blank control group, the Ces1f over expression group, the ALF model group (LPS/D-GalN), and the Ces1f over expression ALF model group. Recombinant adeno-associated virus 8 (rAAV8) containing the Ces1f sequence was injected via the tail vein, while the control group received an injection of the empty viral vector. Three weeks after virus injection, an ALF animal model was established by intraperitoneal injection of LPS+D-GalN. Immunofluorescence was used to observe the transfection efficiency of Ces1f in the mouse liver. Quantitative real-time polymerase chain reaction (qPCR) and Western blotting were employed to determine the expression levels of CES1F, CB1R, and CB2R in mouse liver tissue. Hematoxylin-eosin (H-E) staining was used to observe the pathological changes in the mouse liver tissue. The enzyme-linked immunosorbent assay (ELISA) was utilized to detect the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), arachidonoylethanolamide (AEA), and 2-arachidonoylglycerol (2-AG). Results ·The AAV8 virus had high transfection efficiency and significant hepatotropism in the mouse liver. Compared with the control group, the ALF model group showed decreased mRNA (P<0.001) and protein expression levels of Ces1f and Cb2r (P=0.039, P=0.012). The pathological injury score of liver tissue, the levels of serum ALT, AST, IL-1β, and TNF-α, as well as the levels of Il-1β mRNA, Tnf-α mRNA, AEA, 2-AG, Cb1r mRNA, and CB1R protein in liver tissue were significantly increased (P<0.001). Compared with the ALF model group, the Ces1f over expression ALF model group had significantly increased Ces1f mRNA (P<0.001) and CES1F protein (P=0.002) expression levels, decreased serum AST level (P=0.011), significantly decreased pathological injury scores of liver tissue (P=0.001), decreased levels of serum ALT, IL-1β, and TNF-α, and decreased levels of Il-1β mRNA, Tnf-α mRNA, AEA, and 2-AG in liver tissue (P<0.001). The levels of Cb1r mRNA (P=0.024) and CB1R protein (P=0.027) were also decreased. Conclusion ·Liver-targeted Ces1f gene transfection exerts a protective effect in the ALF mouse model induced by LPS/D-GalN. This protective effect may be achieved by downregulating the levels of hepatic endocannabinoids AEA and 2 - AG and inhibiting the expression of CB1R.

Key words： acute liver failure; carboxylesterase 1f; in vivo liver transfection; endocannabinoid system; mice

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