【作者】 王娜；

【导师】 王凯；

【作者基本信息】 山东大学 ， 内科学（消化系病）， 2016， 硕士

【摘要】 研究背景与目的乙型肝炎病毒(Hepatitis B Virus, HBV)严重威胁人类健康。全球大约有3.5-4.0亿慢性HBV毒携带者。尽管乙型肝炎疫苗的问世,每年仍有5000万例新感染乙型肝炎患者。绝大部分成年的HBV感染者具有自限能力,有5%至10%患者可发展为慢性感染。同时有15%至40%慢性乙型肝炎患者在其一生疾病的进展中将会发生严重的并发症。慢性乙型肝炎(chronic hepatitis B, CHB)感染的自然病史是一个动态过程,根据宿主免疫反应状态的不同可将CHB的自然史分为四个阶段：免疫耐受(Immune tolerant, IT)期,免疫清除(Immune clearance,IC)期,低复制(Low or no-repliicative, LR)期,再活动期；再活动期也被称为HBeAg阴性的慢性乙型肝炎(HBeAg Negative Hepatitis, ENH)。关于乙型肝炎慢性化的具体机制仍不清楚,有效的T细胞应答对病毒的清除起到重要作用,异常免疫应答不利于病毒清除。白细胞介素(Interleukin, IL)-10是一种免疫和炎症抑制因子,可参与Th2型细胞免疫反应。有研究表明IL-10的表达上调与T细胞反应应答抑制有关,并可以导致HBV感染的慢性化。本研究通过检测CHB患者外周血单个核细胞及血浆中IL-10及其相关细胞因子IL-6和肿瘤坏死因子(tumornecrosis factor, TNF)-α的表达水平,进而探讨IL-10在慢性乙型肝炎免疫分期进展中的作用及临床意义。研究方法采用回顾性方法共纳入CHB患者197例,其中免疫耐受期40例、免疫清除期97例、低复制期26例和再活动期34例,选择18例健康志愿者为正常对照。酶联免疫吸附法(Enzyme-linked ImmunosorbentAssay, ELISA)检测每位研究对象血浆IL-10、IL-6和TNF-α水平,实时荧光定量聚合酶链式反应(Real-timePolymerase Chain Reaction, RT-PCR)方法检测每位研究对象外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)和IL-10、IL-6和TNF-α的mRNA水平。采用SPSS22.0进行统计学分析,非正态分布的连续型数据资料以中位数和四分位间距(M[P25,P75])表示；Kmskal-Wallis H检验和Mann-WhitneyU检验用于组间比较。Spearman秩和相关分析用于相关分析。P<0.05时被认为差异具有统计学意义。结果1.CHB患者血浆和PBMCs中IL-10水平较正常对照组明显上调(P<0.05)。我们根据HBeAg和HBVDNA阳性与否分组,分析血浆IL-I0水平和PBMCs IL-10 mRNA水平的表达。HBeAg阳性组患者血浆IL-10水平明显高于HBeAg阴性组(Z=2.591,P=0.01)。而HBVDNA阳性和HBVDNA阴性的CHB患者PBMCs中IL-10 mRNA水平和血浆IL-10水平无明显统计学差异(Z=-1.915,P= 0.099; Z=-1.648,P=0.055)。2.CHB患者血浆和PBMCs中IL-10相关细胞因子IL-6、TNF-α表达水平均也显著高于正常对照组(P<0.05)。CHB患者PBMCs中IL-10 mRNA与IL-6 mRNA水平呈明显正相关(r=0.344,P<0.01),和TNF-α mRNA呈明显负相关(r=0.298, P<0.01)。CHB患者血浆IL-10水平与IL-6蛋白水平呈明显正相关(r=0.514,P<O.01),和血浆TNF-α蛋白呈明显负相关(r=-0.363,P<0.01)。3.CHB患者IL-10 mRNA及蛋白浓度水平与血清ALT呈明显正相关(r=0.392,P<0.01；r=0.264,P<0.01),与血清AST水平呈明显正相关(r=0.290,P<0.01；r=0.184,P=0.01)。4.IL-10蛋白浓度及mRNA水平在不同免疫分期组中有明显统计学意义(P<0.001)。在蛋白水平,免疫清除期患者血浆IL-10水平明显高于免疫耐受期(P<0.05)和低复制期(P<0.05)。在基因水平,免疫清除期PBMCs中IL-10mRNA明显高于免疫耐受期(P<0.001)和低复制期(P<0.05),再活动期PBMCsIL-lOmRNA明显高于免疫耐受期(P=0.01)。Kruskal-Wallis H检验分析IL-6及TNF-α在各免疫分期中没有明显差异。结论免疫清除期患者血浆IL-10水平和PBMCs中IL-10 mRNA水平明显高于免疫耐受期和低复制期患者,再活动期患者PBMCsIL-10 mRNA水平明显高于免疫耐受期。IL-10的表达水平和肝脏的炎症程度密切相关。综上所述,IL-10在慢性乙型肝炎的免疫病程中具有重要作用。更多还原

【Abstract】 Background and ObjectiveHepatitis B virus (HBV) infection is one of the major global public health problems and life-threatening consequences for patients. It is estimated that there are approximately 350-400 million HBV carriers in the world. Even with the advent of an effective hepatitis B vaccine, more than 50 million new cases of HBV infection occurs annually. Although the majority of HBV infected adults recover spontaneously, 5-10% of patients develop chronic HBV infection (carriers, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma), and 15%-40% chronic HBVpatients will develop serious complicantions during their lifetime. It is now recognized that the natural course of chronic hepatits B (CHB) is typically divided into four phases:the immune-tolerant (IT) phase, the immune clearance (IC) phase, low or no-replicative (LR) phase and hepatitis B e antigen (HBeAg)-negative hepatitis (ENH) phase. Although the exact pathophysiology of the development of chronic HBV is not well understood, increasing envidences indicated that effective T-cell responses are crucial for the clearance of viral infection. Aberrant immune response in chronic HBV infection plays a key role in the impairment of HBV clearance. Interleukin-10 (IL-10) is an anti-inflammatory cytokine with a crucial role in preventing inflammatory and regulates the response of T helper type 2 (Th2) cell. Previous studies showed that upregulation of IL-10 were associated with suppressed T-cell responses and chronicity of HBV infection. In our study, we intend to determine the expression of IL-10 and its associated cytokines at gene and protein levels, and then assess the role of IL-10 in the natural history of chronic HBV infection with different immune phases.Patients and MethodsA total of 197 patients with chronic hepatitis B were included, including 40 for immune tolerance (IT) phase,97 immune clearance (IC) phase,26 low replication(LR) phase,34 for HbeAg negative hepatitis B(ENH) phase, as well as 18 healthy controls.The level of IL-10, IL-6 and TNF-α in plasma was determined using enzyme-linked immunosorbent assay (ELISA).The level of IL-10, IL-6 and TNF-α in peripheral blood mononuclear cells (PBMCs) was determined using quantitative real-time polymerase chain reaction(RT-PCR). All statistical analyses were preformed using the IBM SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Data were presented as median values and interquartile ranges (median [centile 25; centile 75]). Comparison between groups was analyzed by the Kruskal-Wallis H analysis. Mann-Whitney U test was used to compare the quantitative variables between two groups.Spearman rank correlation test was used to compare the correlation between variables. Differences were considered statistically significant at a two-tailed P< 0.05.Results1. The plasma levels of IL-10 in chronic hepatitis B patients were significantly higher than healthy controls (all P< 0.05), as well as mRNA levels shared the same condition. Compared IL-10 expression at plasma and mRNA levels stratified by HBeAg and HBVDNA, the results shown that a significant higher level of plasma IL-10 in HBeAg positive patients compared with HBeAg negative patients (Z=-2.591, P=0.01). we did not find any significant difference of IL-10 between CHB patients with HBVDNA positive and those without HBVDNA positive at two levels (Z=-1.915,P= 0.099; Z=-1.648,P= 0.055).2. The levels of IL-10 associated cytokines IL-6 and TNF-α in chronic hepatitis B patients were significantly higher than healthy controls (P< 0.05). IL-10 mRNA in patients with CHB was positively correlated with IL-6 mRNA (r=0.344, P<0.01); whereas negatively correlated with TNF-α mRN A (r=-0.298, P<0.01).Plasma IL-10 in patients with CHB was also positively correlated with plasma IL-6 (r=0.514, P<0.01); whereas negatively correlated with plasmaTNF-α(r=-0.363, P<0.01).3. IL-10 at mRNA and plasma levels in patients with CHB was positively correlated with ALT (r=0.392, P<0.01, r=0.264, P<0.01) and AST(r=0.290, P<0.01; r=0.184,P=0.01)4. There were significant differences of IL-10 at gene and protein levels in the natural history of chronic HBV infection with different immune phases. The expression of IL-10mRNA in IC phase was significantly higher than that in IT phase (P<0.001) or LR phase, whereas we also observe significantly difference of IL-10 mRNA between ITgroup and ENH group (P=0.01). At protein levels, plasma IL-10 in IC phases was significantly higher than IT phase (P< 0.05) or LR phase (P< 0.05). However, we did not find any significant differences of IL-6 and TNF-α within any two of the four phases using Kruskal-Wallis H analysis.ConclusionsThe expression of IL-10 in IC phase was significantly higher than that in IT phase or LR phase at gene and protein levels, whereas we also observe significantly difference of IL-10 mRNA between ITgroup and ENH group.the level of IL-10 expression were associated with the degree of liver inflammation. In summary, IL-10 might contribute to immune phases of chronic HBV infection.更多还原