核苷（酸）类似物在抗乙肝病毒治疗过程中宿主的免疫应答研究

发布时间：2018-03-25 07:11

本文选题：慢性乙型肝炎病毒　切入点：抗病毒治疗　出处：《吉林大学》2014年硕士论文

【摘要】：目的：检测抗病毒治疗过程中慢性乙型肝炎（CHB）患者外周血中的T、B、NK免疫细胞及其血清中细胞因子IL-2, IFN, TNF, IL-4, IL-6和IL-10的表达水平，并与肝脏功能及病毒载量之间进行相关性分析，探讨其对免疫应答的影响。方法：选取2010年9月至2012年10月就诊于吉林大学第一医院门诊及入院的HBeAg阳性CHB患者，签署知情同意后进行筛选试验，其中有54例应用替比夫定初治疗、30例对阿德福韦酯耐药的患者转用恩替卡韦治疗，对照组为20例年龄、性别相匹配的健康人。通过流式细胞技术、微量样本多指标流式蛋白定量技术（Cytometric Bead Array，CBA）检测各组CHB患者外周血中CD3-CD56+,CD244+NK, CD3+, CD3+CD4+, CD3+CD8+, CD4+CD25+Foxp3+, CD4+CD25+CD127low T, CD19+CD27+B细胞以及血清中CD4+T细胞相关的细胞因子（IL-2, IFN, TNF, IL-4, IL-6, IL-10）的含量。应用罗氏公司全自动生化分析仪检测ALT、AST的水平；应用雅培公司化学发光分析仪检测HBsAg、HBsAb、HBeAg、HBeAb；应用荧光定量PCR方法检测HBV DNA病毒载量的变化。分析治疗前后各相应免疫指标与HBV DNA病毒载量、ALT、AST之间的相关性。结果：初始应用替比夫定治疗的HBeAg阳性CHB患者组：(1) CHB患者经替比夫定治疗后，HBV DNA病毒载量、HBsAg乙肝表面抗原定量以及肝功ALT、AST水平较基线时呈下降趋势，治疗3月、6月、9月、13月后，以上各项指标与基线及健康对照组相比，均明显下降（P0.05）;在治疗3月、6月、9月、13月后，分别有5、8、12、15例HBeAg阳性患者出现HBeAg血清学转换;（2）CHB患者经替比夫定治疗后，CD3+CD4+、CD4+CD25+Foxp3+、 CD4+CD25+CD127lowT、CD8+PD-1+T细胞的绝对数呈下降趋势，而CD3-CD56+、CD244+NK、CD3+CD8+T细胞的绝对数以及血清中CD4+T细胞分泌的细胞因子（IL-2, IFN,TNF, IL-4, IL-6，IL-10）的含量呈现上升趋势;（3）CHB患者血清中CD4+T细胞相关的细胞因子（IL-2, IFN, TNF, IL-4, IL-6, IL-10）的水平与HBV DNA病毒载量以及肝功ALT、AST的水平呈明显的负相关。阿德福韦疗效不佳转用恩替卡韦治疗的HBeAg阳性CHB患者组：（1）阿德福韦酯耐药的CHB患者转用恩替卡韦治疗后，HBV DNA病毒载量、HBsAg乙肝表面抗原定量以及肝功ALT、AST水平较基线时呈下降趋势，，治疗3月、6月后，以上各项指标与基线及健康对照组相比，均明显下降（P0.05）;在治疗3月、6月后，分别有4、11例HBeAg阳性患者出现HBeAg血清学转换；（2）阿德福韦酯耐药的CHB患者转用恩替卡韦治疗后，CD3+CD4+、CD4+CD25+Foxp3+、CD4+CD25+CD127lowT、 CD19+CD27+B细胞的绝对数呈下降趋势，而CD3-CD56+、CD244+NK、CD3+CD8+T细胞的绝对数以及血清中CD4+T细胞分泌的细胞因子（IL-2, IFN, TNF, IL-4, IL-6）的含量呈现上升趋势，而IL-10治疗前后无明显变化;（3）阿德福韦酯耐药的CHB患者转换为恩替卡韦治疗后，血清中CD4+IFN+T细胞的水平与HBV DNA病毒载量以及肝功ALT、AST的水平呈明显的负相关。结论:1、CHB患者存在免疫耐受或者免疫功能低下的状态；2、发现在抗病毒治疗后CHB患者免疫细胞及细胞因子与未用药前相比均有显著的升高，表明核苷（酸）类似物可能通过免疫调节起到抑制病毒的作用；3、阿德福韦酯耐药的CHB患者转用恩替卡韦治疗后取得较好的治疗效果，表明恩替卡韦可以作为阿德福韦酯耐药患者的替代治疗；4、在抗病毒治疗前后，CHB患者血清中CD4+T细胞分泌的细胞因子（IL-2, IFN, TNF, IL-4, IL-6、IL-10）的水平与HBV DNA以及肝功ALT、AST的水平具有一定的相关性，表明血清中细胞因子水平与抗病毒治疗后病毒复制情况密切相关；5、CHB患者血清中CD4+T细胞分泌的细胞因子在抗病毒早期可以作为CHB患者治疗过程中的一项临床监测指标，为确定核苷酸类似物治疗疗程提供了新的思路，具有一定的临床意义。
[Abstract]:Objective: chronic hepatitis B detection during antiviral treatment (CHB) in peripheral blood of patients with T, B, NK immune cells and cytokines in the serum of IL-2, IFN, TNF, IL-4, the expression level of IL-6 and IL-10, the correlation analysis between and liver function and viral load, to explore its effect on the immune response.
Methods: from September 2010 to October 2012 in No.1 Hospital of Jilin University from outpatient and hospitalized HBeAg positive CHB patients, informed consent after the screening test, the treatment of 54 cases of patients with early application of telbivudine, 30 cases of en to adefovir resistance of entecavir treatment, 20 cases in the control group age and gender matched healthy people. By flow cytometry, trace sample multivariate flow cytometry quantitative protein technology (Cytometric Bead Array, CBA) detection of CD3-CD56+, CHB in each group in the peripheral blood of patients with CD244+NK, CD3+, CD3+CD4+, CD3+CD8 + CD4+CD25+Foxp3+, CD4+CD25+CD127low, T, CD19+CD27+B cells and CD4+T cells in serum related cytokines (IL-2, IFN, TNF. IL-4, IL-6, IL-10). The contents of application of Roche automatic biochemical analyzer to detect the level of ALT, AST; application of chemiluminescence analyzer HBsA Abbott Company G, HBsAb, HBeAg, HBeAb; fluorescence quantitative PCR method was used to detect the changes of HBV DNA viral load. The correlation between the corresponding immune indexes before treatment and HBV DNA virus load, ALT and AST were analyzed.
Results: the HBeAg positive CHB patients in the initial application of telbivudine treatment: (1) CHB patients after telbivudine treatment, HBV DNA viral load, liver function and HBsAg quantitative hepatitis B surface antigen ALT, AST levels compared to the baseline treatment showed a downward trend, March, June, September, 13 months later, all the above indicators and baseline and compared to healthy controls, were significantly decreased (P0.05); in March June, September, treatment, 13 months later, there were 5,8,12,15 HBeAg positive patients with HBeAg seroconversion; (2) CHB patients treated with telbivudine treatment after CD3+CD4+, CD4+, CD25+Foxp3+, CD4+CD25+CD127lowT, the absolute number of CD8+PD-1+T cells decreased, while CD3-CD56+ CD244+NK, the absolute number of CD3+CD8+T cells and CD4+T cells in serum cytokines (IL-2, IFN, TNF, IL-4, IL-6, IL-10) content showed a rising trend; (3) CD4+T cytokines in the serum of patients with CHB related (I L-2, IFN, TNF, IL-4, IL-6, IL-10) level and HBV DNA viral load and liver ALT was significantly negative correlation between the level of AST.
HBeAg positive patients with CHB A Duff Vee to poor efficacy of entecavir therapy: (1) A Duff Vee ester resistant CHB patients switched to entecavir therapy after HBV DNA viral load, liver function and HBsAg quantitative hepatitis B surface antigen ALT, AST levels compared to the baseline treatment showed a downward trend, March, June, the above indicators with the baseline and compared to healthy controls, were significantly decreased (P0.05); the treatment in March, after June, there were 4,11 HBeAg positive patients with HBeAg seroconversion; (2) A Duff Vee ester resistant CHB patients switched to entecavir after treatment, CD3+CD4+, CD4+CD25+Foxp3+, CD4+CD25+CD127lowT, the absolute number of CD19+CD27+B cells was a downward trend, while CD3-CD56+, CD244+NK, the absolute number of CD3+CD8+T cells and CD4+T cells in serum cytokines (IL-2, IFN, TNF, IL-4, IL-6) content showed a rising trend, and IL-10 before and after treatment No obvious change; (3) patients with CHB conversion of adefovir resistant for entecavir treatment, the serum level of CD4+IFN+T cells and HBV DNA viral load and liver ALT was significantly negative correlation between the level of AST.
Conclusion: 1 CHB patients with immune tolerance or immunocompromised state; 2, found elevated in patients with CHB immune cells and cytokines and not significantly compared to before treatment in antiviral therapy, showed that the nucleoside (acid) analogues may inhibit the virus by immune function; 3, adefovir resistance to en CHB patients better treatment effect achieved after entecavir treatment, showed that entecavir can be used as an alternative treatment of adefovir resistant patients; in 4, before and after antiviral therapy, CD4+T cells secrete cytokines in the serum of patients with CHB (IL-2, IFN, TNF, IL-4, IL-6, IL-10) and HBV and DNA level liver ALT, AST levels have a certain correlation showed that the cytokine level and antiviral treatment in serum is closely related to HBV replication; 5, CD4+T cells secrete cytokines in the serum of patients with CHB In the early stage of antiviral therapy, it can be used as a clinical monitoring index in the treatment of CHB patients. It provides a new idea for determining the treatment course of nucleotide analogues, and has certain clinical significance.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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