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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2023, Vol. 43 Issue (8): 38-51    DOI: 10.13523/j.cb.2304017
研究报告     
LAG-3敲除加重免疫性肝损伤*
万悦1,周斌2,高瑞2,陈慧2,高苗苗2,王效芳3,董典典3,向慎思2,杨晓明1,2,**(),于淼1,2,3,**()
1 安徽医科大学基础医学院 合肥 230032
2 中国人民解放军军事科学院军事医学研究院辐射医学研究所 北京 100850
3 河北大学生命科学学院 保定 071002
LAG-3 Knockout Exacerbates Immune Liver Injury
WAN Yue1,ZHOU Bin2,GAO Rui2,CHEN Hui2,GAO Miao-miao2,WANG Xiao-fang3,DONG Dian-dian3,XIANG Shen-si2,YANG Xiao-ming1,2,**(),YU Miao1,2,3,**()
1 School of Basic Medicine, Anhui Medical University, Hefei 230032, China
2 Beijing Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China
3 School of Life Sciences, Hebei University, Baoding 071002, China
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摘要：

目的:探究淋巴细胞活化基因-3(lymphocyte-activation gene 3, LAG-3)在免疫性肝损伤中的作用及机制。方法:采用尾静脉注射伴刀豆球蛋白A(Concanavalin A, Con A)诱导小鼠急性免疫性肝损伤模型;流式细胞术、实时定量PCR检测肝脏内免疫细胞LAG-3表达;利用LAG-3敲除小鼠研究LAG-3对Con A诱导的免疫性肝损伤的影响,于Con A注射后不同时间点收取外周血,检测血清ALT、AST、细胞因子水平;收集肝脏组织样本进行苏木精-伊红染色(hematoxylin-eosin staining,HE染色)并统计肝脏组织坏死面积;流式细胞术检测肝内细胞分群、T细胞活化;实时定量PCR检测炎症相关基因的表达;酵母双杂交筛选LAG-3相互作用蛋白质。结果:Con A诱导肝脏免疫细胞LAG-3表达增加;LAG-3敲除加重Con A所致小鼠血清转氨酶水平升高、肝脏坏死面积增大、炎症消除延迟;LAG-3 敲除不影响 Con A 诱导的肝内免疫细胞浸润;LAG-3敲除抑制调节性T细胞扩增及IL-10表达;MHCII辅助分子CD74与LAG-3存在蛋白质相互作用。结论:LAG-3敲除加重免疫性肝损伤。
关键词： 免疫性肝损伤;  淋巴细胞活化基因-3;  调节性T细胞;  免疫抑制    
Abstract:

Objective: To investigate the function and mechanism of lymphocyte activation gene 3 (LAG-3) in immune induced liver injury. Methods: Acute immune liver injury model in mice was induced by tail vein injection of concanavalin A (Con A). LAG-3 expression in hepatic immune cells was determined by flow cytometry and real-time PCR. LAG-3 knockout mice were utilized to study the effect of LAG-3 on Con A-induced immune liver injury. Peripheral blood was collected at different time points after Con A injection, and the serum levels of ALT, AST, and cytokines were detected. Liver tissue samples were collected for hematoxylin eosin staining (H & E, hematoxylin eosin staining) and the necrotic area of liver tissue was counted. Intrahepatic immune cell subsets and T cell activation were determined by flow cytometry. The expression of inflammation related genes was detected by real-time quantitative PCR. Yeast two-hybrid screening was performed to find LAG-3 interacting proteins. Results: Con A induced an increase in LAG-3 expression on hepatic immune cells; LAG-3 knockout exacerbated Con A-induced elevation of serum transaminase levels, increased necrotic area of the liver, and delayed resolution of inflammation in mice; LAG-3 knockout did not affect Con A-induced intrahepatic immune cell infiltration; LAG-3 knockout suppressed regulatory T cell expansion and IL-10 expression; MHCII accessory molecule CD74 interacted with LAG-3. Conclusions: LAG-3 knockout aggravates immune liver injury.
Key words: Immune liver injury    Lymphocyte activation gene-3    Regulatory T cells    Immunosuppression
收稿日期: 2023-04-10 出版日期: 2023-09-05
ZTFLH:  Q812  
基金资助: 北京市自然科学基金(7202145)
通讯作者: **电子信箱:xiaomingyang@sina.com; 13718674282@163.com   
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万悦
周斌
高瑞
陈慧
高苗苗
王效芳
董典典
向慎思
杨晓明
于淼

引用本文:

万悦, 周斌, 高瑞, 陈慧, 高苗苗, 王效芳, 董典典, 向慎思, 杨晓明, 于淼. LAG-3敲除加重免疫性肝损伤*[J]. 中国生物工程杂志, 2023, 43(8): 38-51.

WAN Yue, ZHOU Bin, GAO Rui, CHEN Hui, GAO Miao-miao, WANG Xiao-fang, DONG Dian-dian, XIANG Shen-si, YANG Xiao-ming, YU Miao. LAG-3 Knockout Exacerbates Immune Liver Injury. China Biotechnology, 2023, 43(8): 38-51.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2304017    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I8/38

基因名称 上游引物 (5'-3') 下游引物(5'-3')
TBP AGAACAATCCAGACTAGCAGCA GGGAACTTCACATCACAGCTC
IL-2 GTCAACAGCGCACCCACTT TGCTTCCGCTGTAGAGCTTG
IL-4 ACAGGAGAAGGGACGCCAT GAAGCCCTACAGACGAGCTCA
IL-6 GAGGATACCACTCCCAACAGACC AAGTGCATCATCGTTGTTCATACA
IL-10 TGGCCCAGAAATCAAGGAGC CAGCAGACTCAATACACACT
IFN-γ TCAAGTGGCATAGATGTGGAAGAA TGGCTCTGCAGGATTTTCATG
TGF-β CTCCCGTGGCTTCTAGTGC CTCCCGTGGCTTCTAGTGC
LAG-3 CTGGGACTGCTTTGGGAAG GGTTGATGTTGCCAGATAACCC
表1  RT-PCR引物序列
图1  LAG-3+/+小鼠Con A模型,肝脏淋巴细胞表面LAG-3表达增加
图2  LAG-3敲除加重Con A诱导的小鼠肝损伤
图3  LAG-3敲除小鼠Con A模型下肝脏坏死面积增加
图4  LAG-3敲除不影响Con A 诱导的肝内免疫细胞浸润
图5  LAG-3敲除抑制调节性T细胞扩增
图6  LAG-3敲除Con A诱导的血清炎性细胞因子的表达
图7  LAG-3敲除Con A诱导的肝脏炎性细胞因子的表达
图8  酵母双杂交筛选LAG-3相互作用蛋白质
序号 基因名称 NCBI 保存号 选中次数 是否在编码区
1 CD74 NM_001025158.2 6 Y
2 RPL3 NM_000967.3 2 Y
3 FTL NM_000146.3 2 Y
4 RPL29 BC008926.2 2 Y
5 HLA-DRA NM_019111.5 2 Y
6 RPS2 NM_002952 2 Y
7 RPS4X NM_001007.5 2 Y
8 APOO NM_024122.4 1 Y
9 ARL6IP4 NR_103512.1 1 Y
10 C1QC NM_172369.5 1 Y
11 CALM1 NM_006888.4 1 Y
12 CCND3 NM_001760.4 1 Y
13 COMMD4 NM_017828.4 1 Y
14 COX6B1 NM_001863.4 1 Y
15 CYBA NM_000101.3 1 Y
16 EEF1A1 NM_001402.5 1 Y
17 FTH1 NM_002032.2 1 Y
18 GBF1 XM_017016864.2 1 Y
19 GSTO1 NM_001191003.1 1 Y
20 HBA2 NM_000517.4 1 Y
21 HBB NM_000518.4 1 Y
22 HLA-C U41386.1 1 Y
23 IFI27 NM_001288956.1 1 Y
24 IGHV1OR15-1 NR_135694.1 1 Y
25 ITGB2 BC005861.2 1 Y
26 LSP1 NM_002339.2 1 Y
27 MTRNR2L1 NM_001190452.1 1 Y
28 PFN1 NM_005022.3 1 Y
29 PHC2 BC130630.1 1 Y
30 PSME1 NM_001281528.1 1 Y
31 RHOC NM_001042679.1 1 Y
32 RPL19 NM_000981.3 1 Y
33 RPL35 BC000348.2 1 Y
34 RPL36A NM_021029.5 1 Y
35 RPL38 NM_000999.3 1 Y
36 RPL4 NM_000968.3 1 Y
37 RPL7A NM_000972.2 1 Y
38 RPL8 NM_000973.4 1 Y
39 RPS11 NM_001015.4 1 Y
40 RPS14 NM_001025071.1 1 Y
41 RPS3A NM_001006.4 1 Y
42 SELENOP NM_005410.3 1 Y
43 SP140 XM_017003251.2 1 Y
44 SPHK1 NM_001355139.1 1 Y
45 STAB1 NM_015136.2 1 Y
46 STOM NR_073037.1 1 Y
49 SUMF2 XR_002956418.1 1 Y
48 SYNOV2 AK316351.1 1 Y
49 TMSB4X BC016732.1 1 Y
50 TRIM28 NM_005762.2 1 Y
51 ACTG1 BC015779 1 F
52 ARHGDIA XM_011523574.1 1 F
53 CTSD BC016320 1 F
54 DBNL XM_011515325.2 1 F
55 H3F3B NM_005324.4 1 F
56 HLA-E XM_017010809.2 1 F
57 LAPTM5 XM_011542098.2 1 F
58 ND1 MK860679.2 1 F
59 NOL10 BC005125.2 1 F
60 TRIB3 NM_001301201.1 1 F
61 WASHC2C NM_001169107.1 1 F
62 ZNF496 XM_024450375.1 1 F
表2  酵母双杂交筛选的候选LAG-3相互作用蛋白质
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