【摘要】：研究目的 探索慢性乙型肝炎（Chronic hepatitis B，CHB）患者經(jīng)抗病毒治療后HBeAg和HBsAg血清學(xué)轉(zhuǎn)換過程中血清糖蛋白聚糖譜的特征性改變，并探討其生物學(xué)意義。為臨床治療慢乙肝、藥效評價、預(yù)后亦或進(jìn)一步了解慢乙肝的發(fā)病機制提供重要信息。 研究方法 實驗流程見圖1，主要包括以下各部分：（1）對樣本預(yù)處理，，等體積混合4組（正常對照組、eAg+非治療組、E轉(zhuǎn)換治療組和S陰轉(zhuǎn)治療組）病人的血清，利用去氋豐度蛋白試劑盒除去其中的氋豐度蛋白（白蛋白和IgG）；（2）將富集到的低豐度蛋白用Cy5染料進(jìn)行標(biāo)記，并使用分子篩層析除去未標(biāo)記染料；（3）通過凝集素芯片技術(shù)檢測標(biāo)記后的血清蛋白，尋找親和信號的差異。統(tǒng)計學(xué)分析的計量資料采用單向方差分析，事后進(jìn)行Student-Newman-keuls （SNK）兩兩比較。（4）用凝集素印跡驗證芯片結(jié)果。 圖1：技術(shù)路線圖 研究結(jié)果 eAg+非治療組與正常人組相比，對16種凝集素的親和熒光明顯減弱（P0.05），提示在HBV活躍復(fù)制的eAg+非治療組的血清糖蛋白聚糖譜中末端和核心巖藻糖、粘蛋白T/Tn抗原、GalNacα、末端β1-4和β-D鏈接半乳糖、GlcNac、平分型N-乙酰葡糖胺、甘露糖、唾液酸糖鏈結(jié)構(gòu)的明顯降低。E轉(zhuǎn)換治療組與eAg+非治療組相比，包括PSA、MPL和上述16種凝集素對血清糖蛋白聚糖的親和熒光增強（P0.05），說明降低的這些血清糖蛋白聚糖結(jié)構(gòu)又恢復(fù)到或略高于對照組的水平。S陰轉(zhuǎn)治療組較E轉(zhuǎn)換治療組相比，血清糖蛋白聚糖對AAL、ACL、HAL、HPL、RCA-I、LEL、STL、PHA-E、NML和PCL結(jié)合的親和下降到接近對照組（P0.05），示血清糖蛋白中末端巖藻糖、GalNacα、末端β1-4鏈接半乳糖、平分型GlcNAc等結(jié)構(gòu)下降到接近對照組水平；而對VAL、LCA、GNL、PSA、MPL和JAC的親和熒光增強（P0.05），提示末端β-D-半乳糖殘基、核心巖藻糖結(jié)構(gòu)在S陰轉(zhuǎn)治療組增多明顯。 結(jié)論 慢乙肝HBeAg和HBsAg血清學(xué)轉(zhuǎn)換過程中血清糖蛋白聚糖譜發(fā)生了改變，提示血清聚糖變化與慢乙肝HBeAg和HBsAg血清學(xué)轉(zhuǎn)換密切相關(guān)。核心巖藻糖、末端β-D-半乳糖殘基結(jié)構(gòu)可能作為抗HBV治療下HBsAg陰轉(zhuǎn)機制探討相關(guān)的糖標(biāo)志物。 創(chuàng)新點及潛在應(yīng)用價值 1.國內(nèi)外首次對抗病毒治療后慢乙肝HBeAg和HBsAg血清學(xué)轉(zhuǎn)換過程中的人血清糖蛋白進(jìn)行研究。 2.利用高通量的凝集素芯片技術(shù)篩選出了凝集素VAL和LCA親和熒光信號在HBsAg陰轉(zhuǎn)中特異性增強。核心巖藻糖、末端β-D-半乳糖殘基結(jié)構(gòu)可能作為抗HBV治療下HBsAg陰轉(zhuǎn)機制探討的相關(guān)糖標(biāo)志物，為篩選慢乙肝血清學(xué)轉(zhuǎn)換相關(guān)的標(biāo)記物提供信息。
[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 偽, 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 偽, 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).
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R512.62

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相關(guān)期刊論文 前1條

1 馬慧;王江華;郭芳;魏來;;α-2-HS-糖蛋白對聚乙二醇干擾素alfa-2b治療慢性乙型肝炎患者e抗原血清轉(zhuǎn)換預(yù)測作用的研究[J];中國科學(xué):生命科學(xué);2010年09期

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