不同基因型慢性乙型肝炎患者体内黄嘌呤氧化酶和维生素E水平的研究
慢性HBV感染不同临床表型的免疫因子表达模式及免疫遗传特征研究
缩略语表英文缩写英文全称中文名称ALT Alanineaminotransferase 丙氨酸氨基转移酶CCL CC-chemokineligand CC趋化因子配体CXCL CXC-chemokineligand CXC趋化因子配体ELISA Enzyme-linkedimmunoadsordentassay酶联免疫吸附试验FOXP3 Forkhead box P3 叉头型转录因子3GITR Glucocorticoid-Induced TNFR-Relatedprotein 糖皮质激素诱导的肿瘤坏死因子受体相关蛋白GWAS Genome-wide association study 全基因组关联研究HBcAb Hepatitis B core antibody 乙型肝炎核心抗体HBeAg Hepatitis B e antigen 乙型肝炎e抗原HBsAg Hepatitis B surface antigen 乙型肝炎表面抗原HBV HepatitisBvirus 乙型肝炎病毒HCV HepatitisCvirus 丙型肝炎病毒HLA Human leukocyte antigen 人类白细胞抗原IFN Interferon 干扰素IL Interleukin 白细胞介素IRAK1 Interleukin-1 receptor -associated kinase 1白细胞介素1受体相关激酶1 KEGG Kyoto Encyclopedia of Genes andGenomes京都基因与基因组百科全书MHC Majorhistocompatibilitycomplex 主要组织相容性复合物MX1 Myxovirus (influenza virus) resistance 1 粘液病毒抵制子1MyD88 Myeloid differentiation factor 88 髓样分化因子88NF-κB Nuclear factor-kappa B 核因子κBNTCP Sodium/bile acid cotransporter 钠离子/胆酸共转运蛋白RVR Rapid virologic response 快速病毒学应答SNP Single nucleotide polymorphism 单核苷酸多态性SOCS3 Suppressorcytokine signaling 3 细胞因子信号抑制因子3ofSVR Sustained virologic response 持续病毒学应答Tcells 辅助性T细胞Th Helperreceptors Toll样受体TLR Toll-likecell 调节性T细胞Treg RegulatoryTULN Upper limit of normal 正常上限值Cytokine expression pattern and immunogenetic features of different of chronic hepatitis B virus infection patientsAbstractThe mechanism of various clinical states of chronic hepatitis B virus (HBV) infection has been widely concerned. In generally, the individual differences of the host immunity determined by genetic play a crucial role in the clinical outcome of chronic HBV infection. The immune response initiated by the T-cell response to viral antigens is thought to be fundamental for viral clearance and disease pathogenesis in HBV infection. However, there is not clear about the role of numerous cytokines in the mechanism of clinical phenotype diversity of chronic HBV infection.Although the association between HLA-DQ and HLA-DP gene polymorphisms and persistently chronic HBV infection was found by genome-wide association studies, there is no further explanation of the mechanism. There are few study focused on the immune pathogenesis and host genetic pathogenesis of HBeAg-negative chronic hepatitis B. It is necessary to assess the immune state and screen host genetic features for the individual treatment of chronic HBV infection.We aimed to evaluate the immune status by detecting the cytokine/chemokine levels of different clinical phenotypes of chronic HBV infection based on a design protein array, which included 30 cytokines and chemokines associated with anti-HBV immune, to evaluate immune effect of cytokines by protein-protein interactions analysis, which completed through STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), a database of known and predicted protein interactions, to screen the cytokines related to the outcomes of chronic HBV infection on the basis of the protein array results, and to observe the association between gene polymorphisms and clinical phenotype of chronic HBV infection by case-control genetic association studies. The study will enrich the mechanism of clinical phenotype diversity of chronic HBV infection and provide the strategies for predicting the clinical outcome of chronic HBV infection and intervention treatment.The results were listed as follow.1. For HBV infection patients with persistently normal alanine aminotransferase (ALT)levels, the up-regulation of cytokines in JAK-STAT pathway was an important factor. The higher level expression was associated with the better prognosis of HBV infection. The elevation of γ chain cytokines, IL-12 p70, IL-23 p19, and IL-29 in inactive HBV carrier and resolved hepatitis B suggested that the change of lymphocyte function caused by these cytokines may be key factors to promote spontaneous HBeAg seroconversion and HBV clearance.2. For patients with chronic hepatitis B, CXCL9 CXCL10 CXCL11 and CCL20 were available markers to assess the immune state for active hepatitis. The immune foundation of HBeAg-positive chronic hepatitis B patients with ALT over 5 times upper limit of normal was similar to inactive HBV carriers. This may be associated with the high rate of HBeAg seroconversion in this part of patients after antivirus treatment. Compared with HBeAg- positive chronic hepatitis B, the up-regulation expression defection of IL-29, IL-17, IFN- gamma, IL-6, and CCL5 and compensatory increase of IL-21 may be associated with the forming mechanism of HBeAg-negative chronic hepatitis B. Up-regulation of IL-29 and IFN-gamma levels may be used as markers to evaluate serum immunological response for HBeAg-negative chronic hepatitis Bpatients.3. Through genetic association studies between chronic HBV infection and resolved hepatitis B, we demonstrated that gene polymorphisms of natural immunity, inflammation response, and inflammatory chemokines were associated with susceptibility to chronic HBV infection. Among these genes, the most significant was HLA-DQ and HLA-DP gene polymorphisms. HLA-DQ rs7453920, IL10 rs1800872 and MX1 rs467960 were highly linkage, and GTC haplotype with high-risk of chronic HBV infection. TLR9 rs352140 was associated with chronic HBV infection independent of other polymorphisms.4. Through genetic association studies between HBeAg-positive chronic hepatitis B and HBeAg-negative chronic hepatitis B, we found that gene polymorphisms of IL1B, IRAK1, and IL4 were associated with susceptibility to HBeAg-negative chronic hepatitis B. IL12A gene polymorphism for male and IL6R gene polymorphism for female may be associated with susceptibility to HBeAg-negative chronic hepatitis B of male and female, respectively.5. Through genetic association studies between active HBV infection and inactive HBV carriers, we found that gene polymorphisms of IFNG, IL12B, IL15, IL17A, IL1B, IL28B, IL6ST, IL9, and TNFRSF18 were associated with susceptibility to inactive HBV carriers.IFNG rs2069705 was independently associated with inactive HBV carriers and “A” allele was prone to inactive HBV carriers. IL15 rs10833, IL28B rs8099917, IL6ST rs2112979 and TNFRSF18 rs3819001 were highly linkage. CTGC haplotype and TTAC haplotype for female were prone to inactive HBV carries. SLC10A1 rs12882299 for female was associated with inactive HBV carriers.Conclusion: This study comprehensively demonstrated the differences of immune among various states of chronic HBV infection by a protein array and obtained some features associated with HBV clearance or HBeAg seroconversion. The association between inflammatory activities and cytokines levels was further clarified. Through genetic association studies among different states of HBV infection, the gene polymorphisms loci associated with chronic HBV infection, HBeAg-negative chronic hepatitis B, and inactive HBV carriers were screened. This study has enriched the mechanism of various clinical states of chronic HBV infection. Features of immune state and gene polymorphism loci can be used for prognostic assessment of chronic HBV infection.Key words: hepatitis B virus; cellular immunology; protein array; genetic association study; single nucleotide polymorphism慢性HBV感染不同临床表型的免疫因子表达模式及免疫遗传特征研究∗摘要慢性HBV（hepatitis B virus，乙型肝炎病毒）感染多种临床表型的形成机制一直为研究者广为关注。
不同临床类型慢性乙型肝炎HBsAg和HBV DNA水平比较
不同临床类型慢性乙型肝炎HBsAg和HBV DNA水平比较梁志清;吴健林;吴继周;周冬生;唐灵;陶赞英【摘要】目的探讨不同临床类型慢性乙型肝炎(慢性乙肝)对HBsAg和HBV DNA 水平的影响.方法分别采用酶联免疫吸附试验(ELISA)和聚合酶链式反应(PCR)技术检测91例慢性乙肝患者的HBsAg和HBV DNA,并对HBV DNA的检测结果进行对数转换;对不同临床类型测定结果进行统计分析.结果不同临床类型慢性乙型肝炎HBsAg、HBV DNA水平差异均有统计学意义 (P＜0.05),其中慢重肝组与肝硬化组HBsAg和HBV DNA水平比较差异无统计学意义,P值及95%可信区间分别为0.334、-85.05～29.20和0.510、0.59～1.17;慢重肝组HBsAg水平低于慢乙肝组,HBV DNA水平高于慢乙肝组,P值及95%可信区间分别为0.000、163.21～-56.40和0.020、-1.81～-0.16;肝硬化组HbsAg水平低于慢乙肝组,HBV DNA水平高于慢乙肝组,P值及95%可信区间分别为0.006、-139.76～-24.00和-0.005、-2.17～-0.39.结论从HBsAg和HBV DNA水平看,HBsAg和HBV DNA的水平在慢乙肝组的均值显著高于肝硬化组,而肝硬化组又高于慢重肝组;提示慢性乙肝不同临床类型对HBsAg和HBV DNA水平产生一定的影响.【期刊名称】《广西医学》【年(卷),期】2010(032)001【总页数】3页(P15-17)【关键词】慢性乙型肝炎;临床分型;乙型肝炎病毒表面抗原;HBVDNA【作者】梁志清;吴健林;吴继周;周冬生;唐灵;陶赞英【作者单位】桂林医学院附属医院感染性疾病科,桂林市,541001;广西医科大学第一附属医院感染性疾病科,南宁巿,530021;广西医科大学第一附属医院感染性疾病科,南宁巿,530021;桂林医学院附属医院感染性疾病科,桂林市,541001;桂林医学院附属医院感染性疾病科,桂林市,541001;桂林医学院附属医院感染性疾病科,桂林市,541001【正文语种】中文【中图分类】R512.62慢性乙型肝炎(慢性乙肝)的发病机制迄今尚不完全清楚,大多数学者认为,机体的免疫缺陷导致乙肝病毒(HBV)持续感染所引发的免疫损伤是慢乙肝发病的主要原因[1]。
慢性乙型肝炎患者血清补体C3b水平的研究
Ａ ｓ ｔ ｕ ｄ ｙ ｏｎ ｓ ｅ ｒ ｕｍ ｃ ｏ ｍ ｐｌ ｅ ｍｅ ｎｔ Ｃ３ｂ ｌ ｅ ｖ ｅ ｌ ｓ ｏ ｆ ｐ ａ ｔ ｉ ｅ ｎｔ ｓ ｗｉ ｔ ｈ ｃ ｈ ｒ ｏ ｎｉ ｃ ｈ ｅ ｐ ａｔ ｉ ｔ ｉ ｓ Ｂ
Ｊ ｉ ａ ｎ ｇ Ｊ ｉ ｎ ｇ ， Ｌｉ ｕ Ｂｉ ｎ ， Ｒｅ ｙｉ ｈ ａ ｎ Ｘｙ ｉ ｔ ， Ｍｅ ｎ ｇ Ｃ ｕ ｎ ｒ ｅ ｎ
补体 Ｃ ａ ｂ水 平 与 ＨＢ Ｖ 的 感 染 及 肝 细 胞 损 伤 程度 有 关 ， 而 与 病 毒 复 制 的 活 跃 程度 无 关 ， 可 作 为 感 染 和 预 后 的 辅 助检 测 指标 。 关键词 ： 补体 Ｃ ３ ｂ ； 肝 炎病 毒 ， 乙型 ； 丙氨 酸 氨 基 转 氨 酶 类 ； 天 冬 氨 酸 氨 基 转 移 酶 类
ｃ ａ ｎ ｃ ｅ ． Ｍｅ ｔ ｈ ｏ ｄ ｓ １ ４ ０ ｈ ｅ ｐ ａ ｔ ｉ ｔ ｉ ｓ Ｂ ｖ ｉ ｒ ｕ ｓ （ ＨＢＶ） 一 ｉ ｎ ｆ ｅ ｃ ｔ ｅ ｄ ｐ ａ ｔ ｉ ｅ ｎ ｔ ｓ ｗｉ ｔ ｈ ｃ ｈ ｒ ｏ ｎ ｉ ｃ ｈ ｅ ｐ ａ ｔ ｉ ｔ ｉ ｓ Ｂ ｗｅ ｒ ｅ ｅ ｎ ｒ ｏ ｌ ｌ ｅ ｄ ａ ｎ ｄ ｄ ｉ ｖ ｉ ｄ ｅ ｄ ｉ ｎ ｔ ｏ ６ ｇ ｒ ｏ ｕ ｐ ｓ ａ ｃ — ｃ ｏ ｒ ｄ ｉ ｎ ｇ ｔ ｏ ｔ ｈ ｅ ｉ ｒ ｓ ｅ ｒ ｕ ｍ ｌ ｅ ｖ ｅ ｌ ｓ ｏ ｆ ＨＢＶ ｅ ａ ｎ ｔ ｉ ｇ ｅ ｎ （ ＨＢ ｅ Ａｇ ）ａ ｎ ｄ ａ ｌ ａ ｎ ｉ ｎ ｅ ａ ｍｉ ｎ ｏ ｔ ｒ ａ ｎ ｓ ｆ ｅ ｒ ａ ｓ ｅ （ ＡＩ Ｔ）：ｇ ｒ ｏ ｕ ｐ Ａ（ ＨＢ ｅ Ａｇ ：ｐ ｏ ｓ ｉ ｔ ｉ ｖ ｅ ， ＡＩ Ｔ： ｎ ｏ ｒ ｍａ １ ） ， ｇ ｒ ｏ ｕ ｐ Ｂ（ ＨＢ ｅ Ａｇ：ｎ ｅ ｇ ａ ｔ ｉ ｖ ｅ ， ＡＬ Ｔ ：ｎ ｏ ｒ ｍａ １ ） ， ｇ ｒ ｏ ｕ ｐ Ｃ（ ＨＢｅ Ａｇ：ｐ ｏ ｓ ｉ ｔ ｉ ｖ ｅ ， ＡＬ Ｔ ：ｍｉ ｌ ｄ ｌ ｙ ｏ ｒ ｍｏ ｄ ｅ ｒ ａ ｔ ｅ ｌ ｙ ｅ ｌ ｅ ｖ ａ ｔ ｅ ｄ ） ， ｇ ｒ ｏ ｕ ｐ Ｄ （ ＨＢ ｅ Ａｇ：ｐ ｏ ｓ ｉ ｔ ｉ ｖ ｅ ， ＡＬＴ：ｈ ｉ ｇ ｈ ｌ ｙ ｅ ｌ ｅ ｖ ａ ｔ ｅ ｄ ） ， ｇ ｒ ｏ ｕ ｐ Ｅ（ ＨＢ ｅ Ａｇ：ｎ ｅ ｇ ａ ｔ ｉ ｖ ｅ ， ＡＬ Ｔ ：ｍ ｉ ｌ ｄ ｌ ｙ ｏ ｒ ｍｏ ｄ ｅ ｒ ａ ｔ ｅ ｌ ｙ ｅ ｌ ｅ ｖ ａ ｔ ｅ ｄ ） ， ｇ ｒ ｏ ｕ ｐ Ｆ（ ＨＢ ｅ Ａｇ： ｎ ｅ ｇ ａ ｔ ｉ ｖ ｅ ， ＡＬＴ ：ｈ ｉ ｇ ｈ ｌ ｙ ｏ ｒ ｓ ｅ ｖ ｅ ｒ ｅ ｌ ｙ ｅ ｌ ｅ ｖ ａ ｔ ｅ ｄ ） ． Ａｎ ｏ ｔ ｈ ｅ ｒ ２ ０ ｈ ｅ ａ ｌ ｔ ｈ ｙ ｐ ｅ ｏ ｐ ｌ ｅ ｗｅ ｒ ｅ ｓ ｅ ｒ ｖ ｅ ｄ ａ ｓ ｔ ｈ ｅ ｃ ｏ ｎ ｔ ｒ ｏ ｌ ｇ ｒ ｏ ｕ ｐ ． Ｅｎ ｚ ｙ ｍｅ － ｌ ｉ ｎ ｋ ｅ ｄ ｉ ｍｍｕ — ｎ ｏ ｓ ｏ ｒ ｂ ｅ ｎ ｔ ａ ｓ ｓ ａ ｙ （ ＥＬＩ Ｓ Ａ）ｗａ ｓ ｅ ｍｐ ｌ ｏ ｙ ｅ ｄ ｔ ｏ ｄ ｅ ｔ ｅ ｃ ｔ ＨＢ ｅ Ａｇ， ｆ ｌ ｕ ｏ ｒ ｅ ｓ ｃ ｅ ｎ ｃ ｅ ｑ ｕ ａ ｎ ｔ ｉ ｔ ａ ｔ ｉ ｖ ｅ ｐ ｏ ｌ ｙ ｍｅ ｒ ａ ｓ ｅ ｃ ｈ ａ ｉ ｎ ｒ ｅ ａ ｃ ｔ ｉ ｏ ｎ （ Ｐ ＣＲ）ｗａ ｓ ｕ ｓ ｅ ｄ ｔ ｏ
从病毒学角度谈慢性乙型肝炎的功能性治愈(完整版)
从病毒学角度谈慢性乙型肝炎的功能性治愈（完整版）【摘要】通过抗病毒治疗达到慢性乙型肝炎（CHB）的功能性治愈已成为国内外研究的热点和难点，近年来对其临床治愈的标准也基本达成了共识：血清HBV DNA低于检测下限，持续的HBsAg消失，伴或不伴抗-HBs 血清学转换，肝细胞内cccDNA处于非活动转录状态，停止抗病毒治疗后没有疾病复发的情况。
现有的抗病毒治疗方案能有效抑制病毒复制，但难以达到CHB的功能性治愈。
从病毒学方面考虑，乙型肝炎病毒（HBV）cccDNA的转录活性不能得到有效的抑制及病毒基因组DNA的整合导致HBsAg的持续表达是CHB患者功能性治愈率较低的两大根本原因。
此外，HBV独特的复制周期产生的高频突变也会导致表面抗原清除率的降低。
本文将着重围绕病毒学方面的研究现状，探讨功能性治愈CHB的新进展和新策略。
【关键词】肝炎，乙型，慢性；功能性治愈；肝炎病毒，乙型；肝炎表面抗原，乙型；共价闭合环状DNA乙型肝炎病毒（Hepatitis B virus, HBV）是一种威胁全球人类健康的病毒。
根据已公布的统计数据推算，目前我国人群的HBsAg携带率约为6.1%，HBV感染者约8 600万[1]。
慢性乙型肝炎（Chronic hepatitis B，CHB）患者发生肝硬化和肝癌的风险较高，对达到治疗标准的患者进行抗病毒治疗则能减缓疾病的进展并减少肝脏相关疾病的发病风险[2]。
乙型肝炎的功能性治愈是目前国内外公认的抗病毒治疗的理想终点，本文将结合HBV 复制的特点，着重围绕病毒学方面的研究，就CHB功能性治愈的定义、难点及其重要性等问题进行探讨。
一、CHB功能性治愈的定义CHB真正意义上的完全治愈需要彻底清除所有被感染的肝细胞中的共价闭合环状DNA（cccDNA），并且能够在停止抗病毒治疗后不出现因机体失去免疫控制而产生病毒再激活和疾病复发的风险[3]。
现有的抗病毒治疗方案很难做到彻底清除cccDNA，同时cccDNA只存在于被感染的肝细胞中，需要通过有创的肝活检才能加以检测，血清中缺乏明确的生物标志物来反映肝内的cccDNA的水平及其转录活性[4]。
开题报告(慢性乙型肝炎患者血清病毒标志物与cccDNA以及其余肝内病毒标志物的相关性分析)
RNA 转录酶
HBV DNA
HBcAg HBeAg
反转录负链DNA
移至胞浆
合成正链DNA
包含HBV DNA序列上
所有遗传信息的
作为C-mRNA
前基因组RNA(pgRNA)
﹢
HBsAg
S-mRNA
3种亚基因组 RNA
选题依据
理论上cccDNA与HBV DNA、HBsAg、HBeAg、HBcAg应呈正相关
研究内容
病人及肝组织：
2009年3月至2010年6月期间我院住院肝穿的101例病 人，其慢乙肝诊断标准均符合慢性乙型肝炎防治指南。 肝穿组织经固定、脱水、包埋、切片裱片后备用。
血清HBV DNA、HBsAg、HBeAg的测定：
肝穿前后1周内留取血清，检测肝功、HBV DNA、乙 肝五项定量以及AFP、基因型、前C区/BCP区突变。
整合至宿主基因组的整合DNA
cccDNA 3、跨缺口荧光PCR
较好的灵敏度和特异度 石蜡包埋组织即可检测 采用内参校正减少误差 同类方法国外未见报道
2、RCA
不被扩增
扩增
只能扩增 闭合环状DNA
研究方法
肝内cccDNA的检测
1.主要试剂：
石蜡包埋组织切片DNA提取试剂盒(FFPE mini kit, 德国Qiagen公 司)，不降解质粒的ATP依赖的DNA酶(PSAD, EPIBIO公司)， phi29DNA聚合酶(NEB公司)，Taq DNA聚合酶(TaKaRa公司)。
2.方法：
1) 切片脱蜡：石蜡包埋切片二甲苯、梯度酒精脱蜡 2) 提取DNA：FFPE试剂盒提取切片中DNA储存于-40°C冰箱备用 3) PSAD酶消化 (样品DNA8.3ul, PSAD酶0.2ul, ATP应用液0.5ul, 反应缓冲液
慢性乙型肝炎患者血清超氧化物歧化酶的检测以及与干扰素受体表达的关系
慢性乙型肝炎患者血清超氧化物歧化酶的检测以及与干扰素受体表达的关系李国宏;郑风林;刘淑梅【摘要】目的：探讨慢性乙型肝炎患者血清超氧化物歧化酶的检测以及与干扰素受体表达的关系。
方法选取我院2014年1月至2015年1月收治的60例乙型肝炎患者作为观察组，另选60例健康人作为对照组。
两组受试者在实验开始时空腹采集肘静脉血清 SOD、血生化。
随访受试者，督促其在1、3、6、12月复测本研究中所涉及指标，并记录每位受试者病例特点、临床相关危险因素。
结果观察组与对照组比较，血清 SOD 的活力显著降低（P<0.05）；观察组血清 SOD 的活性与血清 ALT 呈现负相关的关系（r =-0.541，P <0.05），SOD 的活性与肝组织病例验证程度呈负相关（r =-0.431，P <0.05）。
结论超氧化物歧化酶是超氧阴离子自由基的特效专一抑制酶，利用清除氧自由基在慢性乙肝中起到抗氧化损伤作用。
缺乏超氧化物歧化酶，可能是造成慢性乙型肝炎患者肝纤维化加重的原因。
对于慢性乙型肝炎患者，在免疫耐受期和病毒低复制期时，超氧化物歧化酶可以抑制外周血单个核细胞干扰素受体αmRNA 的表达。
%Objective:To investigate the serum superoxide dismutase(SOD)of patients with chronic hepatitis b detec-tion and relations with interferon receptor expression. Methods:To choose the hospital between January 2014 and January 2015,60 patients with hepatitis b,as an observer,choose 60 cases of healthy people as the normal control group. At the beginning of the two groups of subjects in the experimental was collected on an empty stomach venous serum SOD,blood bi-ochemistry. Follow-up of subjects,to urge the 1,3,6,12 month indicators involved in the survey of this study,and record each subjectcharacteristics,clinical cases related risk factors. Results:The observation group compared with control group,serum SOD vigor significantly reduced(P < 0. 05);Observation group serum SOD activity and serum ALT present negative correlation relationship(r = 0. 541,P < 0. 05),the activity of SOD and negatively correlated to the degree of liv-er tissue case validation(r = 0. 431,P < 0. 05). Conclusions:Superoxide dismutase(SOD)is a special effects on super-oxide anion inhibit enzyme specificity,using the removal of oxygen free radical oxidation damage effect in chronic hepatitis b. Lack of superoxide dismutase(SOD),may be the cause of chronic hepatitis b patients with liver fibrosis is aggravating. For chronic hepatitis b patients,stage in immune tolerance and low viral replication,superoxide dismutase(SOD)can in-hibit the peripheral blood mononuclear cells(alpha interferon receptor mRNA expression.【期刊名称】《泰山医学院学报》【年(卷),期】2016(037)012【总页数】2页(P1321-1322)【关键词】慢性乙型肝炎;血清超氧化物歧化酶;检测;干扰素受体表达【作者】李国宏;郑风林;刘淑梅【作者单位】德州市市立医院检验科，山东德州253018;德州市市立医院检验科，山东德州 253018;德州市市立医院检验科，山东德州 253018【正文语种】中文【中图分类】R446.1临床数据表明，肝脏疾病患者血液中的超氧化物歧化酶(SOD)、gsh-px、活性均显著低于常人，而抗氧化治疗就能成为治疗各种肝脏疾病的有效途径之一。
不同HBV-DNA载量慢性乙肝患者血清MIF和IL-17的表达意义
不同HBV-DNA载量慢性乙肝患者血清MIF和IL-17的表达意义邹同【摘要】目的探讨不同HBV-DNA载量慢性乙肝患者血清巨噬细胞移动抑制因子(MIF)和白细胞介素-17(IL-17)的表达意义.方法 106例慢性乙肝患者根据HBV-DNA载量分为三组,高载量组29例,中载量组46例,低载量组37例,测定患者血清MIF和IL-17.结果高载量组和中载量组患者血清MIF和IL-17明显高于低载量组(P<0.01),高载量组MIF和IL-17均明显高于中载量组(P<0.05,P<0.01).结论慢性乙肝患者血清MIF、IL-17与病毒载量有密切关系.【期刊名称】《中国实用医药》【年(卷),期】2013(008)009【总页数】2页(P7-8)【关键词】慢性乙肝;病毒载量;巨噬细胞移动抑制因子;白细胞介素-17【作者】邹同【作者单位】453000,河南省新乡市传染病医院【正文语种】中文我国是乙肝病毒感染的高发区，约8% ～20%的患者一旦诊断慢性乙型肝炎病毒感染，经过5年即进展为肝硬化，而失代偿期肝硬化患者预后极差，仅有14% ～35%的5年生存率［1］，因此对于慢性乙肝的治疗非常重要，但是目前多局限在抗病毒的治疗，对于病毒引起的肝脏免疫反应，从而导致肝硬化等方面的治疗进展有限，发现乙肝病毒的免疫反应指导临床治疗尤其重要。
巨噬细胞移动抑制因子(MIF)是一种独特的促肿瘤发生发展的细胞因子已有许多研究，但是其作为炎症介质可以参与机体炎症及免疫反应［2］，而在慢性乙肝的免疫反应有何作用，鲜有报道。
白细胞介素-17(IL-17)在自身免疫性肝病、脂肪肝、及肝脏肿瘤中发挥重要作用，参与了肝脏损伤及再生过程［3］，而是否其也参与到乙肝病毒引起的免疫反应中?本研究探讨慢性乙肝患者不同HBV-DNA载量血清MIF和IL-17的变化。
1 资料与方法1.1 一般资料选择2009年10月至2012年12月本院的慢性乙型肝炎患者，慢性乙肝的诊断依据2010年中华医学会肝病学分会、中华医学会感染病学分会制定指南诊断标准［4］，共106例，其中男61例，女45例，年龄17～63 岁，平均年龄(36.1±8.7)岁。
慢性乙型肝炎患者血清MMP-1、MMP-2、TIMP-1_和HIF-1α_变化及其临床意义探讨
∗基金项目:江苏省卫生健康委员会医学科研项目(编号: JH19060)作者单位:214044江苏省无锡市解放军联勤保障部队第904医院检验科(虞珊珊,张业婷,王元鹏,肖立);南京中医药大学附属无锡医院检验科(袁静文)第一作者:虞珊珊,女,34岁,大学本科,主管技师㊂E-mail: yushanshan3233@通讯作者:肖立,E-mail:xiaoli723723@ ㊃病毒性肝炎㊃慢性乙型肝炎患者血清MMP-1㊁MMP-2㊁TIMP-1和HIF-1α变化及其临床意义探讨∗虞珊珊,张业婷,王元鹏,袁静文,肖立㊀㊀ʌ摘要ɔ㊀目的㊀研究检测慢性乙型肝炎(CHB)患者血清基质金属蛋白酶-1(MMP-1)㊁基质金属蛋白酶-2(MMP-2)㊁基质金属蛋白酶组织抑制因子-1(TIMP-1)和缺氧诱导因子-1α(HIF-1α)水平诊断显著性肝纤维化的效能㊂方法㊀2019年1月~2022年6月我院收治的CHB患者73例和健康体检者50名,采用ELISA法检测血清MMP-1㊁MMP-2㊁TIMP-1和HIF-1α水平,CHB患者接受肝活检,以>=S2为显著性肝纤维化㊂采用受试者工作特征曲线(ROC)分析血清指标诊断肝纤维化的效能㊂结果㊀CHB组血清MMP-1水平为(9.3ʃ2.7)μg/L,显著低于对照组ʌ(12.7ʃ3.3)μg/L,P<0.05ɔ,而血清MMP-2㊁TIMP-1和HIF-1α水平分别为(387.2ʃ54.2)mg/L㊁(296.3ʃ72.9)μg/L和(68.9ʃ11.3)μg/L,均显著高于对照组ʌ分别为(251.6ʃ33.5)mg/L㊁(142.2ʃ23.6)μg/L和(35.1ʃ7.6)μg/L,P<0.05ɔ;经肝穿刺活检组织病理学检查,在73例CHB患者中发现肝纤维化S0~S1期18例㊁S2期22例㊁S3期19例和S4期14例;S4期患者血清MMP-1水平为(6.3ʃ1.8)μg/L,而血清MMP-2㊁TIMP-1和HIF-1α水平分别为(516.7ʃ39.2)mg/L㊁(373.6ʃ55.4)μg/L和(96.8ʃ10.8)μg/L,S3期患者血清MMP-1水平为(7.9ʃ2.2)μg/L,而血清MMP-2㊁TIMP-1和HIF-1α水平分别为(482.5ʃ48.3)mg/L㊁(324.7ʃ59.6)μg/L和(87.5ʃ13.9)μg/L,与S0~S1期或S2期比,差异显著(P<0.05);经ROC分析,血清MMP-1㊁MMP-2㊁TIMP-1和HIF-1α水平诊断CHB患者显著性肝纤维化的截断点分别为10.9μg/L㊁309.4mg/L㊁212.3μg/L和54.1μg/L,其曲线下面积分别为0.835(0.752~0.918)㊁0.948(0.917~0.979)㊁0.955(0.928~0.982)和0.919(0.877~0.961),以血清TIMP-1和HIF-1α水平诊断效能较优㊂结论㊀检测CHB患者血清TIMP-1和HIF-1α或/和MMP-1水平可以帮助筛查显著性肝纤维化,具有一定的临床指导意义㊂㊀㊀ʌ关键词ɔ㊀慢性乙型肝炎;肝纤维化;基质金属蛋白酶;基质金属蛋白酶组织抑制因子;缺氧诱导因子-1α;诊断㊀㊀DOI:10.3969/j.issn.1672-5069.2024.03.003㊀㊀Changes of serum MMP-1,MMP-2,TIMP-1and HIF-1αlevels in patients with chronic hepatitis B㊀Yu Shanshan, Zhang Yeting,Wang Yuanpeng,et al.Department of Clinical Laboratory,904th Hospital,Joint Logistic Support Force,Wuxi 214044,Jiangsu Province,China㊀㊀ʌAbstractɔ㊀Objective㊀This study was to assess the implications of serum matrix metalloproteinase-1(MMP-1),matrix metalloproteinase-2(MMP-2),tissue inhibitor of matrix metalloproteinase-1(TIMP-1)and hypoxia inducible factor-1α(HIF-1α)levels in patients with chronic hepatitis B(CHB).Methods㊀73patients with CHB and50healthy persons were included in this study between January2019and June2022,and serum MMP-1,MMP-2,TIMP-1and HIF-1αlevels were detected by ELISA.All patients with CHB underwent liver biopsies,and the significant liver fibrosis(SLF)was defined as equal to or greater than S2.The diagnostic performance was evaluated by the area under the receiver operating characteristic curve(AUROC).Results㊀Serum MMP-1level in patients with CHB was(9.3ʃ2.7)μg/L,much lower than[(12.7ʃ3.3)μg/L,P<0.05], while serum MMP-2,TIMP-1and HIF-1αlevels were(387.2ʃ54.2)mg/L,(296.3ʃ72.9)μg/L and(68.9ʃ11.3)μg/L,all significantly higher than[(251.6ʃ33.5)mg/L,(142.2ʃ23.6)μg/L and(35.1ʃ7.6)μg/L,respectively,P<0.05]in healthy individuals;the liver histopathological examination showed liver fibrosis S0-S1in18cases,S2in22,S3in19cases and S4in14cases(>=S2in55cases);serum MMP-1level in patients withS4liver fibrosis was(6.3ʃ1.8)μg/L,while serum MMP-2,TIMP-1and HIF-1αlevels were(516.7ʃ39.2)mg/L,(373.6ʃ55.4)μg/L and(96.8ʃ10.8)μg/L,and serum MMP-1level inpatients with S3was(7.9ʃ2.2)μg/L,while serum MMP-2,TIMP-1and HIF-1αlevels were(482.5ʃ48.3)mg/L,(324.7ʃ59.6)μg/L and(87.5ʃ13.9)μg/L,both significantly differentas compared to in patients with S0-S1or with S2(P<0.05);theROC analysis demonstrated that the AUCs were0.835(0.752-0.918),0.948(0.917-0.979),0.955(0.928-0.982)and 0.919(0.877-0.961),when serum MMP-1,MMP-2,TIMP-1and HIF-1αlevels equal to10.9μg/L,309.4mg/L,212.3μg/L and54.1μg/L were set as the cut-off-value in predicting SLF in patients with CHB,superior for serum TIMP-1and HIF-1αto the other two.Conclusion㊀Serum TIMP-1and HIF-1α,and/or MMP-1levels might be used to screen liver fibrosis in patients with CHB,and needs further investigation.㊀㊀ʌKey wordsɔ㊀Hepatitis B;Liver fibrosis;Matrix metalloproteinase;Tissue inhibitor of matrix metalloproteinases;Hypoxia inducible factor-1α;Diagnosis㊀㊀慢性乙型肝炎(Chronic Hepatitis B,CHB)是肝脏感染乙型肝炎病毒(Hepatitis B virus,HBV)所致的慢性肝脏疾病㊂CHB呈慢性进行性发展,可对肝组织结构和功能造成损害㊂肝细胞坏死将导致肝内纤维组织增生,逐渐发展引起肝纤维化和肝硬化,治疗难度大,严重威胁着患者的生命健康㊂因此,及早诊断CHB并评估肝纤维化程度对于临床治疗具有重要的意义[1,2]㊂目前,临床评估肝纤维化的金标准是肝穿刺活检,但其为有创性,在一定程度上限制了该方法的应用㊂因此,探寻评估肝纤维化的无创诊断方法仍是当今研究的重要问题[3,4]㊂肝纤维化的发生机制是细胞外基质(extracellular matrix,ECM)的产生和降解不平衡,进而引起间质胶原和其他成分过度沉积[5]㊂基质金属蛋白酶(matrix metallopro-teinases,MMPs)和基质金属蛋白酶组织抑制因子(tissue inhibitor of matrix metalloproteinases,TIMPs)对ECM的产生和降解具有调节作用㊂近些年来,随着对MMPs和TIMPs相关研究的深入,应用它们诊断肝纤维化和肝硬化也取得了显著的成果[6,7]㊂本研究检测了CHB患者血清MMP-1㊁MMP-2㊁TIMP-1和缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)水平,分析了以上指标诊断肝纤维化的效能,现报道如下㊂1㊀资料与方法1.1一般资料㊀2019年1月~2022年6月我院收治的CHB患者73例,男50例,女23例;年龄为28~ 67岁,平均年龄为(38.5ʃ5.6)岁㊂符合‘慢性乙型肝炎防治指南“[8]的诊断标准㊂排除标准:(1)酒精性肝病㊁自身免疫性肝病;(2)合并肺纤维化㊁心肌梗塞㊁肿瘤及其他可影响血清MMPs和TIMPs水平的疾病;(3)妊娠期或哺乳期妇女;(4)入组前患者已接受免疫调节剂或抗病毒治疗㊂另选择同期在本院体检的健康人50例作为对照组,男30例㊁女20例;年龄为32~65岁,平均年龄为(37.7ʃ6.8)岁㊂受试者签署知情同意书,本研究已获得我院医学伦理委员会批准㊂1.2肝活检㊀在超声引导下,使用美国巴德MG-1522型全自动活检枪和16G活检针行快速肝穿刺活检,取得1.5~2.5cm长的肝组织,立刻置入10%中性福尔马林溶液中固定,脱水㊁石蜡包埋㊁制作4μm的切片,分别进行HE㊁Masson和网状纤维染色,评估肝纤维化程度分期㊂1.3血清检测㊀采用ELISA法检测血清MMP-1㊁MMP-2㊁TIMP-1(上海赛培森生物科技有限公司)和HIF-1α水平(上海化邦生物科技有限公司)㊂1.4统计学方法㊀应用SPSS26.0软件进行统计学分析,先应用Shapiro-Wilk进行正态性检验,对符合正态分布的计量资料以(xʃs)表示,采用t检验;计数资料以%表示,采用x2检验或Fisher精确概率计算;应用ROC曲线分析血清MMP-1㊁MMP-2㊁TIMP -1和HIF-1α诊断CHB患者显著性肝纤维化的效能㊂P<0.05为差异有统计学意义㊂2㊀结果2.1两组血清MMP-1㊁MMP-2㊁TIMP-1和HIF-1α水平比较㊀CHB组血清MMP-1水平显著低于健康人组(P<0.05),而血清MMP-2㊁TIMP-1和HIF-1α水平显著高于健康人组(P<0.05,表1)㊂表1㊀两组血清MMP-1㊁MMP-2㊁TIMP-1和HIF-1α水平(xʃs)比较例数MMP-1(μg/L)MMP-2(mg/L)TIMP-1(μg/L)HIF-1α(μg/L) CHB739.3ʃ2.7①387.2ʃ54.2①296.3ʃ72.9①68.9ʃ11.3①健康人5012.7ʃ3.3251.6ʃ33.5142.2ʃ23.635.1ʃ7.6㊀㊀与对照组比,①P<0.052.2不同肝纤维化分期的CHB 患者血清MMP -1㊁MMP -2㊁TIMP -1和HIF -1α水平比较㊀经肝穿刺活检组织病理学检查,在73例CHB 患者中发现肝纤维化S0~S1期18例㊁S2期22例㊁S3期19例和S4期14例;检测发现,随着肝纤维化分期的加重,CHB 患者血清MMP -1水平逐渐降低,而血清MMP -2㊁TIMP -1和HIF -1α水平逐渐升高(P <0.05,表2)㊂2.3血清MMP -1㊁MMP -2㊁TIMP -1和HIF -1α水平诊断CHB 患者显著性肝纤维化的效能分析㊀经ROC 分析发现,血清TIMP -1和HIF -1α水平诊断CHB 患者显著性肝纤维化的效能优于其他两个指标(P <0.05,表3㊁图1)㊂表2㊀不同肝纤维化分期患者血清指标(x ʃs )比较例数MMP -1(μg /L)MMP -2(mg /L)TIMP -1(μg /L)HIF -1α(μg /L)S0~S11812.4ʃ2.9288.3ʃ34.1203.7ʃ47.945.2ʃ8.8S22210.5ʃ2.4①351.9ʃ31.5①229.4ʃ38.7①61.3ʃ10.2①S3197.9ʃ2.2①②482.5ʃ48.3①②324.7ʃ59.6①②87.5ʃ13.9①②S4146.3ʃ1.8①②③516.7ʃ39.2①②③373.6ʃ55.4①②③96.8ʃ10.8①②③㊀㊀与S0~S1相比,①P <0.05;与S2相比,②P <0.05;与S3相比,③P <0.05表3㊀血清MMP -1㊁MMP -2㊁TIMP -1和HIF -1α水平诊断显著性肝纤维化的ROC 分析截断点AUC 95%CI 标准误P 敏感度特异性MMP -110.9μg /L0.8350.752~0.9180.0420.0000.7140.731MMP -2309.4mg /L 0.9480.917~0.9790.0180.0000.8860.860TIMP -1212.3μg /L 0.9550.928~0.9820.0160.0000.9150.826HIF -1α54.1μg /L 0.9190.877~0.9610.0230.0000.9300.817图1㊀血清MMP -1㊁MMP -2㊁TIMP -1和HIF -1α水平诊断显著性肝纤维化的ROC 曲线3㊀讨论肝纤维化是慢性肝炎发展至肝硬化或肝癌过程中的重要环节,也是其必经的病理学发展阶段㊂与肝硬化相比,肝纤维化病情较轻㊂如果患者早期得到及时的治疗,肝纤维化仍可逆转㊂及早判定CHB 患者肝纤维化程度将有利于临床针对患者病情制定合理的治疗方案,从而改善患者预后[9,10]㊂研究显示[11,12],在慢性肝纤维化的发生发展过程中MMPs 和TIMPs 等多种细胞因子起到缓解或促进作用,能够作为评估肝纤维化的参考指标㊂本研究检测血清MMP -1㊁MMP -2㊁TIMP -1和HIF -1α水平,旨在探究其在CHB 患者的临床意义㊂本研究CHB 组血清MMP -1水平显著低于健康人,并且随着肝纤维化分期的加重,血清MMP -1水平逐渐降低㊂成纤维细胞㊁肝星状细胞㊁肝细胞是肝组织MMP -1的主要来源㊂患者发生肝纤维化时,MMP -1活性出现相对或绝对的降低,其原因可能是发生肝纤维化后,转化生长因子-β(transforming growth factor -β,TGF -β)和TIMP -1等物质抑制了MMP -1的表达㊂研究显示,TGF -β能够降低MMP -1mRNA 表达,而增强TIMP -1mRNA 表达[13]㊂本研究还发现CHB 组血清MMP -2和TIMP -1水平显著高于健康人,并且随着肝纤维化程度的加重,血清MMP -2和TIMP -1水平逐渐升高,提示血清MMP -2和TIMP -1水平与肝纤维化的发展存在一定的相关性㊂MMP -2能够降解基底膜中的Ⅳ型胶原支架,导致功能性基底膜的完整性受损,进而引起肝星状细胞生存的微环境改变,导致肝星状细胞被激活,间质性胶原和Ⅰ型胶原沉积增多,引起基质金属蛋白酶Ⅰ型表达提高,促进MMP -2酶原激活和MMP -2水平升高,进而导致窦周基质继续降解,刺激未转化的肝星状细胞活化,引起细胞外基质分泌增加,形成恶性循环㊂因此,在肝纤维化形成过程中MMP -2具有重要的作用[14,15]㊂作为TIMPs 家族分布最广泛的成员之一,TIMP-1能够抑制MMPs及其酶原活性㊂另外,TIMP-1能抑制聚集的细胞外基质降解,抑制肝星状细胞凋亡,在肝纤维化的发生发展过程中起到重要作用[16]㊂研究显示[17],肝纤维化组织TIMP-1表达显著升高,因而TIMP-1可作为评估肝纤维化的参考指标㊂本研究CHB组血清HIF-1α水平显著高于健康人组,并且随着肝纤维化分期的加重,血清HIF-1α水平显著升高,提示血清HIF-1α水平与肝纤维化的发生发展存在一定的相关性㊂HIF-1α作为一种转录调控因子,可参与机体调节氧代谢的过程㊂近些年,有报道指出肝纤维化的发病机制与HIF-1α具有密切的联系㊂国外研究通过动物实验发现HIF -1α水平在肝纤维化的小鼠肝脏组织显著上调,并且肝脏组织的纤维化程度与血清HIF-1α水平存在显著的正相关性[18]㊂HIF-1α对肝纤维化的促进作用可能是通过激活TGF-β信号通路实现的㊂使用TGF-β1型受体激酶抑制剂处理肝纤维化小鼠可抑制TGF-β信号通路激活,减轻肝纤维化程度,减少细胞外基质沉积[19]㊂肝纤维化的发生发展伴有新生血管的大量形成,而新生血管与血管内皮生长因子(vascular endothelial growth factor,VEGF)水平上调有关,其作为HIF-1α的下游基因,VEGF表达水平受HIF-1α的影响可显著升高㊂因此,HIF-1α可上调VEGF水平促进肝纤维化的发生[20]㊂ʌ参考文献ɔ[1]Tan M,Bhadoria AS,Cui F,et al.Estimating the proportion ofpeople with chronic hepatitis B virus infection eligible for hepatitis B antiviral treatment worldwide:a 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