抗HBV血清学转换中血清糖蛋白聚糖谱的变化及其临床意义

发布时间：2018-07-31 15:28

【摘要】：研究目的探索慢性乙型肝炎（Chronic hepatitis B，CHB）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。为临床治疗慢乙肝、药效评价、预后亦或进一步了解慢乙肝的发病机制提供重要信息。研究方法实验流程见图1，主要包括以下各部分：（1）对样本预处理，，等体积混合4组（正常对照组、eAg+非治疗组、E转换治疗组和S阴转治疗组）病人的血清，利用去氋丰度蛋白试剂盒除去其中的氋丰度蛋白（白蛋白和IgG）；（2）将富集到的低丰度蛋白用Cy5染料进行标记，并使用分子筛层析除去未标记染料；（3）通过凝集素芯片技术检测标记后的血清蛋白，寻找亲和信号的差异。统计学分析的计量资料采用单向方差分析，事后进行Student-Newman-keuls （SNK）两两比较。（4）用凝集素印迹验证芯片结果。图1：技术路线图研究结果 eAg+非治疗组与正常人组相比，对16种凝集素的亲和荧光明显减弱（P0.05），提示在HBV活跃复制的eAg+非治疗组的血清糖蛋白聚糖谱中末端和核心岩藻糖、粘蛋白T/Tn抗原、GalNacα、末端β1-4和β-D链接半乳糖、GlcNac、平分型N-乙酰葡糖胺、甘露糖、唾液酸糖链结构的明显降低。E转换治疗组与eAg+非治疗组相比，包括PSA、MPL和上述16种凝集素对血清糖蛋白聚糖的亲和荧光增强（P0.05），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的水平。S阴转治疗组较E转换治疗组相比，血清糖蛋白聚糖对AAL、ACL、HAL、HPL、RCA-I、LEL、STL、PHA-E、NML和PCL结合的亲和下降到接近对照组（P0.05），示血清糖蛋白中末端岩藻糖、GalNacα、末端β1-4链接半乳糖、平分型GlcNAc等结构下降到接近对照组水平；而对VAL、LCA、GNL、PSA、MPL和JAC的亲和荧光增强（P0.05），提示末端β-D-半乳糖残基、核心岩藻糖结构在S阴转治疗组增多明显。结论慢乙肝HBeAg和HBsAg血清学转换过程中血清糖蛋白聚糖谱发生了改变，提示血清聚糖变化与慢乙肝HBeAg和HBsAg血清学转换密切相关。核心岩藻糖、末端β-D-半乳糖残基结构可能作为抗HBV治疗下HBsAg阴转机制探讨相关的糖标志物。创新点及潜在应用价值 1.国内外首次对抗病毒治疗后慢乙肝HBeAg和HBsAg血清学转换过程中的人血清糖蛋白进行研究。 2.利用高通量的凝集素芯片技术筛选出了凝集素VAL和LCA亲和荧光信号在HBsAg阴转中特异性增强。核心岩藻糖、末端β-D-半乳糖残基结构可能作为抗HBV治疗下HBsAg阴转机制探讨的相关糖标志物，为筛选慢乙肝血清学转换相关的标记物提供信息。
[Abstract]:Objective to investigate the characteristic changes of serum glycoproteoglycan profile in patients with chronic hepatitis B (Chronic hepatitis) treated with antiviral therapy during serological conversion between HBeAg and HBsAg, and to explore its biological significance. To provide important information for clinical treatment of CHB, evaluation of drug efficacy, prognosis or further understanding of the pathogenesis of CHB. Methods the experimental procedure is shown in figure 1, which includes the following parts: (1) the serum samples were pretreated, and the serum samples were mixed in 4 groups (normal control group, non-treatment group, E conversion group and S negative group). The enriched low abundance proteins (albumin and IgG); (2) were labeled with Cy5 dyes and the unlabeled dyes were removed by molecular sieve chromatography. (3) Lectin chip technique was used to detect the labeled serum protein and to find the difference of affinity signal. The measurement data of statistical analysis were analyzed by one-way ANOVA and compared with each other by Student-Newman-keuls (SNK). (4) Lectin blotting was used to verify the results of the chip. Figure 1: technical roadmap study results: the eAg non-treatment group compared with the normal group, The affinity fluorescence of 16 lectins decreased significantly (P0.05), suggesting that in the serum glycoproteoglycan profile of the active replication of HBV eAg, the terminal and core fucose, the mucin T/Tn antigen GalNac 伪, the terminal 尾 1-4 and 尾 -D linked galactosamine GlcNac. Mannose, sialic acid chain structure significantly decreased. E conversion treatment group compared with eAg non-treatment group, The affinity fluorescence of PSA-MPL and 16 lectins to serum glycoproteoglycan was enhanced (P0.05), which indicated that the reduced structure of serum glycoproteoglycan returned to or slightly higher than that of the control group. The binding affinity of serum glycoproteoglycan to RCA-ILEL PCL of HPLL of AALL ACLL HALL decreased to that of the control group (P0.05), which indicated that the structures of the serum glycoprotein, such as GalNac 伪, 尾 1-4 linked galactose and GlcNAc, were reduced to the level of the control group. However, the affinity fluorescence of JAC and PSAMPL was enhanced (P0.05), suggesting that the terminal 尾 -Dgalactose residue and core fucose structure increased significantly in S-negative group. Conclusion the changes of serum glycoproteoglycan profiles during serological conversion between HBeAg and HBsAg suggest that the changes of serum glycosaminoglycans are closely related to serological changes of HBeAg and HBsAg. The structure of core fucose and terminal 尾 -Dgalactose residues may be used to explore the mechanism of HBsAg negative transition in HBV treatment. Innovation and potential application value 1. The study of human serum glycoprotein during serological conversion of HBeAg and HBsAg after the first antiviral therapy at home and abroad. 2. High throughput lectin chip technology was used to screen agglutinin VAL and LCA affinity fluorescence signals. The structure of core fucose and terminal 尾 -Dgalactose residues may be used as a related sugar marker to explore the mechanism of HBsAg negative transformation under HBV treatment, and provide information for screening markers related to serological transformation of chronic hepatitis B (CHB).
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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