抗HBV治疗后血清学转换中血清糖蛋白聚糖谱的变化及其临床意义

Alteration of the glycan profile of serum glycoproteins during the seroconversion process in hepatitis B virus-infected patients treated with antiviral therapy and its clinical significance

目的 探索慢性乙型肝炎患者经抗病毒治疗后HBeAg和HBsAg血清学转换过程中血清糖蛋白聚糖谱的特征性改变,并探讨其生物学意义.方法 在去除高丰度蛋白质的基础上,应用凝集素芯片技术比较分析正常对照组、HBeAg阳性非治疗组、HBeAg转换治疗组、HBsAg阴转治疗组的血清糖蛋白聚糖亲和谱,并用凝集素印迹方法验证芯片结果.计量资料采用单因素方差分析,进而进行Student-Newman-keuls （SNK）法两两比较.结果 HBeAg阳性非治疗组与正常对照组相比,对16种凝集素的亲和荧光明显减弱（P值均＜0.05）,提示在HBV活跃复制的HBeAg阳性非治疗组患者的血清糖蛋白聚糖谱中末端和核心岩藻糖、黏蛋白T/Tn抗原、α-N乙酰半乳糖胺（GalNac α）、末端β 1-4和β-D链接半乳糖、N-乙酰葡糖胺（GlcNac）、平分型GlcNac、甘露糖、唾液酸糖链结构明显降低.HBeAg转换治疗组与HBeAg阳性非治疗组相比,包括豌豆凝集素、桑橙凝集素和上述16种凝集素对血清糖蛋白聚糖的亲和荧光增强（P值均＜ 0.05）,说明降低的这些血清糖蛋白聚糖结构又恢复到或略高于对照组水平.HBsAg阴转治疗组与HBeAg转换治疗组相比,血清糖蛋白聚糖对橙黄网孢盘菌凝集素、尾穗苋凝集素、螺旋蜗牛凝集素、螺旋圆口螺凝集素、蓖麻凝集素、番茄凝集素、茄块茎凝集素、红腰豆凝集素、眼镜蛇罗非鱼凝集素和红花菜豆凝集素结合的亲和下降,接近对照组（P值均＜0.05）,提示血清糖蛋白中末端岩藻糖、GalNacα、末端β 1-4链接半乳糖、平分型GlcNac等结构下降到接近对照组水平;而对凝集素槲寄生凝集素、扁豆凝集素、雪花莲凝集素、豌豆凝集素、桑橙凝集素和木菠萝凝集素的亲和荧光增强（P＜ 0.05）,提示末端β-D-半乳糖残基、核心岩藻糖结构在HBsAg阴转治疗组明显增多.结论 血清糖蛋白聚糖变化与HBeAg和HBsAg血清学转换密切相关.核心岩藻糖、末端β-D-半乳糖残基结构可作为抗HBV治疗后HBsAg阴转相关的糖标志物.

Objective To use a lectin microarray to study the alteration of glycan affinity profiles of serum glycoproteins during the hepatitis B e antigen （HBeAg） and hepatitis B surface antigen （HBsAg） seroconversion in patients with chronic hepatitis B （CHB） following treatment with antiviral therapy,and to explore its biological significance.Methods CHB patients were divided into the following four groups：untreated HBeAg-positive,HBeAg seroconversion after anti-HBV therapy,HBsAg loss after anti-HBV therapy,and healthy individuals （controls）.Serum samples were collected from each participant,depleted of high abundance proteins and analyzed by a lectin microarray containing 50 lectins.The lectin-affinity glycan profiles of serum proteins were partially verified by lectin blotting.Between-group differences were analyzed by one-way analysis of variance,and pairwise comparisons were carried out with the Student-Newman-Keuls （SNK） method.Results The results from the lectin microarray and lectin blotting assay showed significantly reduced affinity for 16 lectins in the untreated HBeAg-positive group compared to the control group （P〈0.05）;in addition,the specific glycan profiles of the untreated HBeAg-positive group included decreased terminal and core fructose,GalNAc α-Thr/Ser （T,Tn-antigen）,GalNac α,terminal β1-4,and β-D galactose,bisecting and/or GlcNAc,mannose and Sia.However,the HBeAg seroconversion after anti-HBV therapy group showed enhanced binding of PSA,MPL and the above-mentioned 16 lectins （P 〈 0.05）,suggesting that the reduced serum glycoprotein glycan structures returned to normal or slightly higher than healthy levels after the therapy-induced seroconversion.Comparison of the group with HBsAg loss after anti-HBV therapy to the group with HBeAg seroconversion after anti-HBV therapy showed the binding ability of ten lectins （AAL,ACL,HAL,HPL,RCA-I,LEL,STL,PHA-E,NML and PCL） were weakened to near control levels and six lectins （VAL,LCA,GNL,PSA,MPL and JAC） were significantly strengthened （all P 〈 0.05）.These findings implied that the glycan containing terminal fructose,GalNacα,terminal β1-4 galactose,and bisecting GlcNAc glycan structures dropped to near control levels,while the terminal β-D-galactose residues and core fructose structure increased significantly.Conclusion The glycan structures of serum glycoproteins are closely related to HBeAg and HBsAg seroconversion in CHB patients.It is possible that a special lectin binding glycan involving the terminal β-D-galactose residues and core fructose may act as sugar markers associated with the disappearance of serum HBsAg during anti-HBV therapy for CHB.