趋化因子IP-10及自然杀伤细胞在病毒性肝炎抗病毒治疗过程中作用的研究

发布时间：2018-02-26 10:26

本文关键词： 乙型丙型肝炎慢性 IP-10 NK细胞　出处：《吉林大学》2013年博士论文　论文类型：学位论文

【摘要】：乙型肝炎病毒(Hepatitis B virus，HBV)和丙型肝炎病毒(Hepatitis C virus，HCV)感染是导致肝炎、肝硬化、肝癌非常重要的危险因素。目前我国大概有9300万的乙肝感染者和600~1000万丙肝感染者。宿主的免疫因素在乙型肝炎及丙型肝炎的易感性、疾病的发生发展、对治疗的反应等方面发挥着重要作用。宿主固有免疫中的NK细胞在慢性乙型肝炎的发生发展及趋化因子IP-10的血浆学表达及其单核苷酸多态性在慢性丙型肝炎的易感性及对抗病毒治疗应答均发挥着重要的作用。本实验将阐述宿主免疫因素对慢性乙型肝炎及慢性丙型肝炎的发病机制及对抗病毒治疗疗效的影响。趋化因子是一类具有化学趋化活性的细胞因子，在炎症反应中起重要作用。大量研究证实：趋化因子的异常表达与病毒性肝炎疾病的进程密切相关。高表达的趋化因子可招募大量效应淋巴细胞如细胞毒性T淋巴细胞至肝内以清除病毒感染的肝细胞，但同时由于趋化因子的过度释放，，导致炎症反应扩大，加剧了肝细胞损伤。因此明确相关趋化因子在病毒性肝炎中的作用机制尤为重要。 IP-10是1985年发现的趋化因子CXC亚族成员，在病毒性肝炎的发生发展及肝脏的免疫损伤中扮演着重要角色。多项研究显示，在慢性丙型肝炎患者肝脏及血浆中检测到高水平IP-10的表达。其高表达与肝脏炎症活动程度和纤维化密切相关。目前，IP-10的单核苷酸多态性与血浆学表达与HCV感染后的结局及干扰素治疗后应答的关系还不清楚。 NK细胞是天然免疫细胞，在抗病毒免疫中发挥重要作用。NK细胞可以直接杀伤病毒感染的细胞并能间接通过抗体介导发挥细胞介导的细胞毒作用。NK细胞的功能取决于其活化性受体和抑制性受体与不同种类细胞表面的配体相结合。这些配体包括典型的和非典型的MHC I类抗原，MHC样蛋白和其他各种自身和病毒衍生的分子。它们可能组成形表达或表达于病毒感染的细胞表面。在HBV感染的过程及应用核苷酸类似物抗病毒治疗过程中NK细胞所起到的作用及变化鲜有报道。因此本研究第一部分采用质谱技术和酶联免疫吸附测定法等技术：检测慢性丙型肝炎患者、感染慢性丙型肝炎病毒后自发清除者及健康对照者中血浆IP-10表达水平的差异；观察慢性丙型肝炎患者在干扰素联合利巴韦林抗病毒治疗前、抗病毒治疗中及抗病毒治疗后患者血浆IP-10表达的变化；结合慢性丙型肝炎患者抗病毒治疗的疗效，找到能预测疗效的最佳的IP-10表达阈值；在慢性丙型肝炎患者、自发清除者及健康对照者中对IP-10的SNP位点rs3921；rs8878；rs4859584；rs4241578；rs4859588；rs56061981；rs74810361；rs4256246进行测序分析，分析IP-10不同的SNP位点的不同表型与慢性丙型肝炎感染及抗病毒治疗后疗效的关系。第一部分实验结果如下：HCV自发清除者血浆IP-10表达比慢性丙型肝炎患者及健康对照者低，同时健康对照者比慢性丙型肝炎患者血浆IP-10表达低；用于治疗慢性丙型肝炎患者的干扰素和利巴韦林会诱使血浆IP-10表达增高，但治疗结束后24周时血浆IP-10表达比未治疗前明显降低；在干扰素联合利巴韦林抗病毒治疗前基线IP-10表达小于600pg/ml是患者容易获得完全早期病毒学应答的独立预测因素；在干扰素联合利巴韦林抗病毒治疗后2周时IP-10表达小于540pg/ml是患者容易获得早期病毒学应答的独立预测因素；IP-10基因中所检测的8个SNP位点与慢性丙型肝炎病毒易感性无关，与抗病毒疗效无关。第二部分采用流式细胞技术观察替诺福韦组及阿德福韦酯组患者的抗病毒疗效；检测慢性乙型肝炎患者外周血中NK细胞数量及受体表达与正常对照者之间的差异；观察替诺福韦组及阿德福韦酯组在抗病毒治疗过程中NK细胞受体表达的变化情况，并比较两者间的差异；找到可能预测慢性乙型肝炎患者抗病毒疗效的NK细胞受体。第二部分实验结果如下：替诺福韦和阿德福韦酯治疗的乙肝患者具有不同的抗病毒疗效，替诺福韦抗病毒疗效优于阿德福韦酯；慢性乙型肝炎患者肝脏损伤与NK细胞受体表达的改变有关；应用替诺福韦或阿德福韦酯抗病毒治疗可以降低慢性乙型肝炎患者NK细胞的抑制性受体NKG2A~+和KIR2DL3~+的表达；NKG2A~+和KIR2DL3~+受体的表达可能是临床上评价慢性乙型肝炎抗病毒疗效的预测因子。综上所述，IP-10在慢性丙型肝炎感染及抗病毒治疗过程中发挥着重要的作用，并且IP-10表达的高低可直接预测患者抗病毒的早期疗效；NK细胞在慢性乙型肝炎感染过程中发挥着重要作用，并且NKG2A~+和KIR2DL3~+受体的表达可能是临床上评价慢性乙型肝炎抗病毒疗效的预测因子。
[Abstract]:Hepatitis B virus (Hepatitis B, virus, HBV) and hepatitis C virus (Hepatitis C, virus, HCV) infection is the leading cause of hepatitis, liver cirrhosis, liver cancer risk factors is very important. At present there are about 93 million of hepatitis B infection and HCV infection. 600~1000 host immune factors in hepatitis B and C the occurrence and development of disease susceptibility, and plays an important role in the response to treatment and so on. All play an important role in host innate immunity in NK cells, susceptibility and response to antiviral treatment and expression of single nucleotide polymorphism in chronic hepatitis C in patients with chronic hepatitis B of the occurrence and development of chemokine IP-10 in plasma. The implications of this experiment will explain the pathogenesis and host immune factors of efficacy of antiviral therapy of chronic hepatitis B and chronic hepatitis C.
Chemokines are chemotactic cytokines activity, plays an important role in inflammatory reaction. Many studies have confirmed that abnormal expression of hepatitis B virus disease of chemotactic factors closely related to the process. The high expression of chemokines can recruit a large number of effector cells such as cell toxicity to the liver in T lymphocytes remove the virus infected liver cells, but also because of the excessive release of chemokines, leading to inflammation expansion, exacerbated liver injury. Therefore clarify the mechanism of chemokines in viral hepatitis is particularly important.
IP-10 is found in 1985 of CXC chemokine subfamily members, plays an important role in the occurrence and development of liver immune injury and viral hepatitis. The studies show that high levels of IP-10 expression detection in patients with chronic hepatitis C in liver and plasma. Its high expression is closely related with the severity of inflammation and liver fibrosis. At present, the polymorphism of IP-10 and plasma science and outcomes and the relationship between the expression of response to interferon therapy after HCV infection is not clear.
NK cells play an important role in innate immune cells,.NK cells can directly kill virus infected cells in antiviral immunity and antibody mediated indirectly through the function of.NK cells play a cytotoxic effect on the cell mediated activation of receptor and inhibition with ligand cell surface receptors. The combination of these ligands include typical and atypical MHC class I antigen molecules, MHC like protein and various other autoimmune and virus derived. They may form or expression on the surface of infected cells.
There are few reports on the role and changes of NK cells in the process of HBV infection and the application of nucleotide analogues to antiviral therapy.
So the first part of this study by mass spectrometry and enzyme-linked immunosorbent assay technique: detection of patients with chronic hepatitis C, chronic hepatitis C virus infection after spontaneous clearance of the differential expression of plasma IP-10 in patients and healthy controls; observation of patients with chronic hepatitis C in interferon and ribavirin treatment, the expression changes of IP-10 in plasma in patients with antiviral the treatment and antiviral therapy; combined antiviral curative effect in the treatment of patients with chronic hepatitis C, found to predict the efficacy of the best IP-10 expression threshold; in patients with chronic hepatitis C, SNP locus rs3921 spontaneous clearance of IP-10 patients and healthy controls; rs8878; rs4859584; rs4241578; rs4859588; rs56061981; rs74810361; rs4256246 by sequencing analysis, analysis of different phenotypes in patients with chronic hepatitis C infection of different sites and IP-10 SNP The relationship between antiviral treatment and curative effect.
The first part of the experiment results are as follows: the spontaneous clearance of HCV plasma IP-10 expression than in patients with chronic hepatitis C and healthy controls were low, while healthy controls were lower than the expression in plasma of patients with chronic hepatitis C IP-10; for the treatment of patients with chronic hepatitis C interferon and Leigh Bhave Lin will induce the expression of plasma IP-10 increased, but 24 weeks after the end of treatment the plasma IP-10 the expression was significantly lower than before treatment; interferon combined with antiviral therapy in Leigh Bhave Lin before the baseline expression of IP-10 is less than 600pg/ml with easy access to complete early virological response independent predictive factors; interferon combined with antiviral therapy in Leigh Bhave Lin 2 weeks after the expression of IP-10 is less than 540pg/ml were the independent predictors to obtain early virological response to 8 SNP; in patients with chronic hepatitis C virus susceptibility loci detected in the IP-10 gene, and antiviral The curative effect was not related.
The second part of the study for the antiviral efficacy of tenofovir group and adefovir dipivoxil group with flow cytometry; the difference between the expression quantity and detection of NK cell receptor in patients with chronic hepatitis B in peripheral blood and normal controls; observe the tenofovir group and adefovir dipivoxil group changes in expression of NK cell receptor antiviral treatment process. And compare the differences between the two; find the NK cell receptor may predict the efficacy in patients with chronic hepatitis B virus.
The second part of the experimental results are as follows: for patients with hepatitis B and tenofovir, adefovir dipivoxil treatment with antiviral efficacy of different, tenofovir antiviral efficacy than adefovir dipivoxil; expression in patients with chronic hepatitis B and injury of NK cell receptor changes; expression of tenofovir or adefovir dipivoxil antiviral therapy can reduce NK cells in patients with chronic hepatitis B the inhibitory receptor NKG2A~+ and KIR2DL3~+; the expression of NKG2A~+ and KIR2DL3~+ receptor may be a predictor of curative effect evaluation of chronic hepatitis B patients.
In summary, IP-10 plays an important role in chronic hepatitis C infection and antiviral treatment, early treatment effect and the expression level of IP-10 can be directly predicted with the virus; NK cells play an important role in chronic hepatitis B infection, and the expression of NKG2A~ + and KIR2DL3~+ receptor may be a predictor of curative effect evaluation of chronic hepatitis B antiviral therapy in clinic.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

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