羧酸酯酶1f基因活体肝转染对急性肝衰竭影响的研究

doi:10.3969/j.issn.1674-8115.2025.11.007

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (11): 1480-1489.doi: 10.3969/j.issn.1674-8115.2025.11.007

• 论著 · 基础研究 • 上一篇

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¹, 于倩¹, 张钰婷¹, 李玎嘉¹, 张乔婷², 刘亮明¹^,³()

^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

收稿日期:2025-02-20 接受日期:2025-07-03 出版日期:2025-11-28 发布日期:2025-12-03
通讯作者: 刘亮明，教授、主任医师，博士；电子信箱：liuliangming@shsmu.edu.cn。
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)

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

BIAN Shu¹, YU Qian¹, ZHANG Yuting¹, LI Dingjia¹, ZHANG Qiaoting², LIU Liangming¹^,³()

^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

Received:2025-02-20 Accepted:2025-07-03 Online:2025-11-28 Published:2025-12-03
Contact: LIU Liangming, E-mail: liuliangming@shsmu.edu.cn.
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)

摘要/Abstract

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

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

中图分类号:

R575.3

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.

BIAN Shu, YU Qian, ZHANG Yuting, LI Dingjia, ZHANG Qiaoting, LIU Liangming. Research on the impact of in vivo liver transfection of carboxylesterase 1f gene on acute liver failure[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(11): 1480-1489.

图/表 8

图1 载体结构图谱Note： The objective gene is located in the pAV-TBG-P2A-GFP vector. The TBG promoter drives the expression of the target gene. The Kozak sequence GCCACC is located upstream of the target gene. A flag tag is added to the C-terminus of the target gene. The target gene and GFP are expressed in a non-fusion manner through the P2A sequence. Moreover, there are ASIS I/MluI restriction sites at both ends of the gene.

Fig 1 Schematic diagram of the vector structure

表1 Real-time PCR基因名称及引物序列

Tab 1 Primer sequences for qPCR

Gene	Primer	Sequence (5′→3′)	Product/bp
Ces1f	F	TGGAGAGTCAGCAGGAGGTTACAG	290
	R	AGCACACTTGTCACGAACTGGTC	290
Cb1r	F	GCCTTGCAGATACCACCTT	103
	R	TTGAGCCCACGTAGAGGA	103
Cb2r	F	GAAAGCCCTCGTACCTGTT	121
	R	GGTCACACTGCCGATCTT	121
Il-1β	F	AGTTGACGGACCCCAAA	104
	R	TCTTGTTGATGTGCTGCTG	104
Tnf-a	F	CGCTGAGGTCAATCTGC	110
	R	GGCTGGGTAGAGAATGGA	110
Gapdh	F	GACATGCCGCCTGGAGAAAC	244
	R	GTCCACCACCCTGTTGCTGTAG	244

图2 AAV8转染小鼠肝脏且具有肝趋向性Note： A. Fluorescence microscopy images of liver tissue sections from each group of mice (×400). B. Fluorescence microscopy images of tissue sections from various organs of mice (×400). GFP: Green fluorescent protein, with fluorescence signals appearing green; DAPI: Nuclear dye, with fluorescence signals appearing blue. Scale bar=150 μm.

Fig 2 AAV8 transfection in mouse liver demonstrates liver tropism

图3 各组小鼠肝组织 Ces1f 基因和蛋白质的表达情况Note： A. Statistical histogram of Ces1f mRNA expression. B. CES1F protein expression. The left panel shows the Western blotting image, and the right panel displays the statistical histogram of relative protein expression levels (n=5).

Fig 3 Expression of Ces1f gene and protein in liver tissues of mice from each group

图4 各组小鼠血清转氨酶水平和肝组织病理及评分Note： A. Statistical histograms of serum transaminase levels in mice from each group. The left panel shows the statistical histogram of ALT levels, and the right panel displays the statistical histogram of AST levels. B. Liver pathology and scores in mice from each group. The left panel presents H-E staining images of liver tissues (×200), and the right panel shows the statistical histogram of histological scores for liver injury severity (n=5). Scale bar=150 μm.

Fig 4 Serum transaminase levels and liver pathology with scoring in mice from each group

图5 各组小鼠炎症因子TNF-α和IL-1β水平变化情况Note： A. Changes in serum levels of inflammatory factors TNF-α and IL-1β in mice. The upper panel shows the statistical histogram of serum IL-1β levels, and the lower panel displays the statistical histogram of TNF-α levels. B. Changes in mRNA levels of inflammatory factors Tnf-α and Il-1β in liver tissues of mice. The upper panel presents the statistical histogram of Il-1β mRNA expression levels, and the lower panel shows the statistical histogram of Tnf-α mRNA expression levels (n=5).

Fig 5 Changes in the levels of inflammatory factors TNF-α and IL-1β in each group of mice

图6 各组小鼠肝组织AEA和2-AG水平变化情况Note： A. Statistical histogram of AEA expression levels in liver tissues of mice. B. Statistical histogram of 2-AG expression levels in liver tissues of mice (n=5).

Fig 6 Changes in the levels of AEA and 2-AG in the liver tissues of each group of mice

图7 各组小鼠肝组织 Cb1r 和 Cb2r 基因和蛋白质的表达情况Note： A. Protein expression of CB1R and CB2R. The left panel shows the Western blotting image, the upper right panel displays the statistical histogram of relative CB1R protein expression levels, and the lower right panel presents the statistical histogram of relative CB2R protein expression levels. B. The mRNA expression of Cb1r and Cb2r. The upper panel shows the statistical histogram of relative Cb1r mRNA expression levels, and the lower panel displays the statistical histogram of relative Cb2r mRNA expression levels (n=5).

Fig 7 Expression of Cb1r and Cb2r genes and proteins in the liver tissues of each group of mice

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羧酸酯酶1f基因活体肝转染对急性肝衰竭影响的研究

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

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