半乳糖上调内皮细胞Siglec-9聚糖配体水平抑制巨噬细胞活性  

Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity

作　　者：吴妮婷 摄渊婷 刘红梅[1] 李瑾[1] 贾乙[1] WU Niting;SHE Yuanting;LIU Hongmei;LI Jin;JIA Yi(Department of Pharmaceutics,Faculty of Pharmacy and Laboratory Medicine,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)药学与检验医学系药剂学教研室,重庆400038

出　　处：《陆军军医大学学报》2024年第13期1502-1511,共10页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(81973319)。

摘　　要：目的探究半乳糖对人原代肺动脉内皮细胞上唾液酸特异性Ig样凝集素-9(sialic-acid-binding immunoglobulin-like lectin 9,Siglec-9)聚糖配体表达的影响及作用机制。方法采用流式细胞术确定内皮细胞表面Siglec-9聚糖配体的表达并鉴定该聚糖配体末端糖苷键类型;分别用左旋葡萄糖、葡萄糖、N-乙酰基葡萄糖、甘露糖、N-乙酰基甘露糖、半乳糖、唾液酸、蔗糖处理内皮细胞48 h,采用流式细胞术检测内皮细胞Siglec-9聚糖配体的表达;将内皮细胞分为对照组和半乳糖组(n=3),Western blot、流式细胞术、免疫荧光分析半乳糖对内皮细胞Siglec-9聚糖配体表达的影响;α2-3,6,8唾液酸酶处理内皮细胞后,Western blot检测半乳糖对内皮细胞Siglec-9聚糖配体恢复时间的影响;半乳糖处理内皮细胞后,Western blot检测分析细胞内唾液酸合成关键酶(GNE、NANS、NANP、CMAS、NPL以及ST3Gals)表达水平,并用RT-qPCR验证相关蛋白的mRNA表达;siRNA敲低NANP、CMAS及ST3Gal-Ⅲ,Western blot检测内皮细胞Siglec-9聚糖配体水平变化;巨噬细胞与内皮细胞共培养实验分析Siglec-9聚糖配体的增加对巨噬细胞的凋亡与吞噬能力的影响。结果内皮细胞上表达有Siglec-9聚糖配体且该配体为末端结构为α2-3连接的唾液酸糖蛋白;与对照组比较,半乳糖组内皮细胞上该聚糖配体表达水平显著增加(P<0.01),但半乳糖的添加对该配体的自我恢复时间无影响;与对照组比较,半乳糖处理的内皮细胞中NANP、CMAS、ST3Gal-Ⅲ表达水平升高(P<0.05,P<0.01),且RT-qPCR验证结果与其一致;抑制内皮细胞中NANP、CMAS、ST3Gal-Ⅲ的表达后,内皮细胞Siglec-9聚糖配体水平下降(P<0.01)。共培养实验中,与未处理组比较,半乳糖处理的内皮细胞促进巨噬细胞凋亡增加(P<0.01),并导致其吞噬能力减弱(P<0.05)。结论半乳糖通过NANP-CMAS-ST3Gal-Ⅲ通路上调内皮细胞上Siglec-9聚糖配体水平,从而抑制巨噬细胞�Objective To investigate the effect and mechanism of galactose on the expression of sialic-acid-binding immunoglobulin-like lectin 9(Siglec-9)glycan ligand on human primarily cultured pulmonary artery endothelial cells.Methods The expression of Siglec-9 glycan ligand on the endothelial cell surface and the type of glycosidic bonds at the end of this ligand were identified by flow cytometry.Endothelial cells were treated with L-glucose,glucose,N-acetylglucose,mannose,N-acetylmannose,galactose,sialic acid and sucrose for 48 h,and the expression of Siglec-9 glycan ligand in endothelial cells was detected by flow cytometry.The endothelial cells were divided into the control group and the galactose group(n=3).Western blotting,flow cytometry,and immunofluorescence assay were employed to investigate the impact of galactose on the Siglec-9 glycan ligand in endothelial cells.Afterα2-3,6,8 sialidase treatment for endothelial cells,Western blotting was used to detected the effect of galactose on the recovery time of Siglec-9 glycan ligand in endothelial cells.Endothelial cells were treated with galactose,Western blotting was used to detect and analyze the expression levels of intracellular sialic acid synthetases(GNE,NANS,NANP,CMAS,NPL and ST3Gals),and the mRNA expression levels of the relevant proteins were verified by RT-qPCR.Western blotting was used to detect the changes of Siglec-9 glycan ligand level in endothelial cells after siRNA knockdown in NANP,CMAS and ST3Gal-Ⅲ.The effect of the increase of Siglec-9 ligand on apoptosis and phagocytosis of macrophages was analyzed by macrophage co-culture experiments.Results Endothelial cells showed the expression of Siglec-9 ligand and the ligand was a sialic acid glycoprotein linked toα2-3 sialic acid at the end.Compared with the control group,the expression level of this ligand on endothelial cells in the galactose group was increased significantly(P<0.01),but the addition of galactose had no effect on the self-recovery time of the ligand.Compared with the control group,t

关 键 词：半乳糖 内皮细胞 唾液酸特异性Ig样凝集素-9聚糖配体 唾液酸合成关键酶 巨噬细胞 

分 类 号：R343.23[医药卫生—基础医学] R322.12R329.25