慢加急性乙型肝炎肝衰竭单核巨噬细胞TLR-4糖基化改变及调控机制研究

The sialylated and N-glycolyl-Sia glycans were more highly expressed in ACHBLF than in CHB and HC by lecin microarray and mass spectrometric analysis in our study. The abnormal expression of TLR-4 induced by endotoxin lipopolysaccharide (LPS) was associations with the mononuclear macrophage （M/MΦ）paralyzed in acute-on-chronic hepatitis B liver failure (ACHBLF) patients at the same time（the research foundation）. It was known that LPS-induced NF-κB activation was dependent on TLR4 regulated by the Neu1 activity associated with LPS-stimulated M/MΦ. The precisely information about galactosylated modification of TLR-4 on ACHBLF M/MΦ was unknown. To identify N- and O- linked with glycosylation and glycan of TLR-4 on M/MΦ of ACHBLF patients by magnetic particles and mass spectrometric analysis. The glycans of TLR-4 were confirmed by lectins microarray and glycosylation sites mutation. The role of TLR-4 in M/MΦ paralyzed was clarified. The data provided pivotal information from the cell immune functions and glycomics respectively to clarify the pathogenesis of ACHBLF.

申请者通过凝集素芯片及质谱技术发现慢加急性乙型肝炎肝衰竭（ACHBLF）患者血清唾液酸型糖链及N-羟乙酰基-唾液酸表达显著升高，同时发现ACHBLF患者单核巨噬细胞（M/MΦ ）失能与内源性内毒素(LPS)诱导的Toll样受体-4（TLR-4）异常表达相关（工作基础），既往研究证实LPS诱导激活TLR-4-NF-κB信号通路受唾液酸酶Neu1 正向调控，目前ACHBLF患者TLR-4糖基化修饰未见报道。本研究拟以ACHBLF单核巨噬细胞TLR-4为研究对象，通过磁性微粒复合物分离及MALDI-TOF-MS质谱等技术鉴定TLR-4的N及O-糖基化位点，凝集素芯片及糖基化位点突变筛选验证特异糖链，确定Neu1调控TLR-4糖链，观察TLR-4糖基化变化及特异性糖链与单核巨噬细胞失能相关性，结合免疫学及糖组学方法探讨ACHBLF发病机制。

本研究通过糖组学及免疫学研究寻找慢加急性乙肝肝衰竭（ACHBLF）患者糖链变化与免疫功能内在联系。凝集素芯片联合MALDI-TOF-MS质谱分析检测ACHBLF患者血清糖型变化， ACHBLF与CHB及HC相比，识别GalNAcα/ β1-3 /6Gal的WFA，识别GlcNAc及αGal的GSL-II 、GSL-I， 识别Gal的PTL-II， 识别分支型 (LacNAc)n的 PWM 以及识别Galβ1-3GalNAc，GalNAc末端的BPL均明显增加， EEL、LTL及ACA等较HC组显著降低；识别Galβ-1,4GlcNAc (II型)、Galβ1-3GlcNAc (I型) 的ECA，识别Galβ1-3GalNAc, GalNAc的MPL，识别αGalNAc，Tn抗原，GalNAcα1- 3(Fucα1-2)Gal的DBA，以及识别高甘露糖、Manα1-6Man的NPA较CHB明显升高(≥ 2.33， p≤ 0.005) ， 3 种糖链特异性出现ACHBLF患者 (m/z 2158.942， 2590.262， 2603.237) ，凝集素Blot表现出了良好的重现性，主要为岩澡糖、唾液酸型糖链及GalNAc及复合Gal 糖链，同时木糖（xylose ，xyl) 和 N-羟乙酰基-唾液酸高表达，PHA-E+L芯片结果和组化结果一致显示平分型GlcNAc和双天线N-糖链在ACHBLF肝脏细胞高表达， ACHBLF患者单核巨噬细胞膜上TLR4表达与预后相关， TLR-4-NF-κB 信号通路受唾液酸酶Neu1调控，依赖Neu1调控使连接到TLR-4的β-半乳糖苷去除α-2,3-唾液酸残基，通过该研究证实ACHBLF患者血清唾液酸糖链改变与LPS激活的TLR-4-NF-κB信号通路直接相关，首次揭示了ACHBLF 患者特有的糖蛋白糖链对单核巨噬细胞功能影响。