基于抗体多维数据的解析探讨丙型肝炎病毒抗体亲和力在病程中的动力学变化及与HLA多态性的关联

发布时间：2018-06-21 23:12

本文选题：丙型肝炎病毒 + 抗体亲和力　；参考：《大连医科大学》2016年博士论文

【摘要】：背景:丙型肝炎病毒(Hepatitis C Virus,HCV)是输血后以及散发性肝炎的主要病原,是世界性的公共卫生难题。全世界约有1亿7千万HCV血清学阳性的患者,面临着转变成肝硬化、肝癌的风险。尽管HCV能刺激机体产生有效的免疫反应,但80%的HCV感染者会进展成慢性丙肝患者,只有20%患者能有效清除病毒,自行恢复且不留任何后遗症。大多数HCV慢性感染患者体内能产生针对不同HCV病毒蛋白的抗体,而且在患者血清中能检测到这些抗体的存在。显然,HCV感染的宿主免疫反应的类型和强度可能对其预后用着重要作用。HCV抗体检测对HCV诊断有重要价值,中和抗体能直接作用于HCV病毒,但一般认为通常在HCV感染者中所检测到的抗体是针对HCr43蛋白(HCV非结构区NS3及核心区的编码产物)、c100-3抗原(HCV非结构区NS3、NS4的编码产物)的抗体,为非中和抗体,因此,不能通过检测HCV抗体准确判定HCV感染者的免疫状态。我们在对乙型肝炎anti-HBc亲和力与HBV感染的联系中发现同为非中和抗体,但anti-HBc亲和力与HBV感染的预后关系密切;也有研究证明抗体亲和力高低能准确反映巨细胞病毒感染的活动状况。测定HCV抗体亲和力有可能较为准确的反映HCV感染者的免疫状况,对判定HCV感染的现状和结局提供有价值的信息。抗体的总活性取决于抗体蛋白的含量及其与抗原间的结合活性(亲和力)。而目前临床实验室hcv抗体的检测只检测抗体的总活性,并未涉及抗体蛋白的含量和抗体亲和力的检测,本研究将hcv抗体总活性解析成抗体蛋白量和抗体亲和力,试图利用常规的hcv抗体总活性的检测系统,参照乙型肝炎anti-hbc亲和力的检测原理来建立hcv抗体亲和力的检测方法,并探讨丙型肝炎病毒抗体亲和力、总活性及物质量与病毒载量、机体损伤程度以及疾病不同阶段变化的动力学联系。近十年对hla和hcv感染持续性及清除的研究非常多,机体对病毒表位的免疫反应取决于抗原递呈细胞加工和递呈抗原表位的能力,以及免疫细胞识别这些抗原表位的能力,而这些功能是由hla分子遗传决定的,因而推测hla类型可影响hcv感染结局。hla-i类等位基因与hcv感染的自限性转归之间存在很强的相关性,例如hla-i类等位基因a3、b27和cw*01均与病毒清除相关,而b8则与病毒的持续性感染相关。ns3区域的一个表位对hla-dr具有高度亲和性,提示hla-Ⅱ类分子也与病毒清除有关。某些hla-Ⅱ类等位基因也与hcv感染的结局相关,与病毒清除最为高度相关的hla-Ⅱ类等位基因是dqb1*0301基因和drb1*1101。然而,这些研究结果并不完全一致,甚至相互矛盾。目的:采用抗体多维数据分析,建立hcv抗体亲和力的检测方法,并探讨丙型肝炎病毒抗体亲和力、物质量及总活性动力学变化及其与病毒量、机体损伤程度以及疾病不同阶段的联系。hcv感染患者hla-a,hla-b,hla-drb1基因型别与疾病过程的联系以及其作用机制。方法:1、采用重组乙型肝炎疫苗反复三次皮下注射免疫兔子,第一次注射一周后采集兔子血清,获得低亲和力抗体动物血清;第三次注射两周后采集兔子血清,获得高亲和力抗体动物血清,倍比稀释血清,采用双抗原夹心酶联免疫吸附实验测定anti-hbs。随机收集临床诊断为hcv感染患者血清106例,采用化学发光微粒子免疫检测法测定hcv抗体,pcr-荧光探针法检测hcv-rna,同时检测alt、ast、alb等临床常用检测指标。根据alt、ast水平以及hcv-rna水平各分三组,比较分析组间抗体亲和力、抗体总活性及抗体物质量的变化。2、随机收集临床诊断为hcv感染患者血清328例,采用化学发光微粒子免疫检测法测定hcv抗体,pcr-荧光探针法检测hcv-rna,同时检测alb。根据疾病阶段分为慢性肝炎组和肝硬化组,慢性肝炎组又根据患者alb和hcv-rna水平分为:alb正常hcv-rna阴性组(好转恢复阶段)和其他组(进展阶段)。分析比较组间抗体亲和力、抗体物质量及抗体总活性的变化。3、随机收集临床诊断为hcv感染患者血清104例及健康对照200例,采用化学发光微粒子免疫检测法测定hcv抗体,pcr-荧光探针法检测hcv-rna,同时检测血清alb水平,采用测序方法(sequencebasedtyping,pcr-sbt)进行hla基因型的鉴定。比较分析不同hla型别间alb水平、抗体亲和力、抗体物质量及抗体总活性的变化。4、采用网上搜索数据库结合discoverystudio(ds)软件来分析预测ns3和hla-drb1*09,hla-a*02,hla-a*33,hla-b*58,hla-drb1*07,hla-drb1*13这6种hla蛋白分子之间的相互作用。数据库包括美国国立医学图书馆文献检索系统ncbi,蛋白质结构数据库pdb以及prosite数据库。结果:1、雌兔第1次免疫后血清抗体亲和力为0.522,第2次为0.826,第3次为0.896;雄兔第1次免疫后血清抗体亲和力为0.816,第2次为0.846,第3次为0.972,均呈逐渐升高趋势,与抗体亲和力成熟规律一致。2、alt、ast正常组的抗体物质量明显低于任一异常组、均异常组(p0.05),而抗体亲和力则明显高于任一异常组、均异常组(p0.05)。抗体总活性在三组中比较,均没有显著性差异(p0.05)。3、根据hcv-rna水平,将alt、ast均正常组(53例)进一步分组为hcv-rna阴性组和hcv-rna阳性组。hcv-rna阴性组的抗体总活性、抗体物质量均明显低于hcv-rna阳性组(p0.05),而抗体亲和力却明显高于hcv-rna阳性组(p0.05)。4、hcv-rna阴性组的抗体总活性、抗体物质量均明显低于低病毒载量、高病毒载量组(p0.05),而抗体亲和力则明显高于低病毒载量、高病毒载量组(p0.05)。5、以hcv-rna阳性为因变量,抗体亲和力、抗体物质量和抗体总活性为自变量,进行logistic回归分析,hcv-rna水平与抗体亲和力呈负相关(p0.05)。6、alb正常hcv-rna阴性组的抗体亲和力明显高于慢性肝炎其他组和肝硬化组(p0.05),而抗体物质量明显低于慢性肝炎其他组和肝硬化组(p0.05),抗体总活性在三组中比较没有显著性差异(p0.05)。7、抗体亲和力和抗体物质量的roc曲线下面积分别是0.816、0.811,均显示了较高的诊断效能,明显高于传统的抗体检测(roc曲线下面积为0.581)。抗体亲和力对于区分丙肝感染进展阶段(hcv-rna阳性)和好转阶段(hcv-rna阴性)的最佳诊断临界点为0.873,抗体亲和力越高,病毒复制越少。8、alb正常hcv-rna阴性组中,高抗体亲和力(≥0.873)的比例明显高于慢性肝炎其他组和肝硬化组(p0.05)。9、hcv-rna(+)的血清被含有高亲和力抗体的血清中和后,lgrna明显低于中和前的水平(p0.05)。10、在hcv患者和健康对照组中鉴定出8种hla型别具有显著性差异(p0.05),分别是hla-a*02,hla-b*39,hla-drb1*09,hla-drb1*11,hla-a*33,hla-b*58,hla-drb1*07,hla-drb1*13。其中hla-a*02,hla-b*39,hla-drb1*09,hla-drb1*11在hcv患者中阳性率明显低于健康对照组,而hla-a*33,hla-b*58,hla-drb1*07,hla-drb1*13则明显高于健康对照组(p0.05),提示hla-a*02,hla-b*39,hla-drb1*09,hla-drb1*11为丙肝感染的抵抗基因,而hla-a*33,hla-b*58,hla-drb1*07,hla-drb1*13则是丙肝感染的敏感基因。11、在上述8种hla型别中,血清alb水平在hla-a*02,hla-a*33,hla-b*39,hla-drb1*07,hla-drb1*11,hla-drb1*13基因携带者与非携带者间并无差异(p0.05),仅hla-b*58和hla-drb1*09携带者与非携带者的alb水平明显不同,前者p=0.015,后者p=0.003,均0.05,但仅有hla-drb1*09在交叉验证中仍然具有显著性差异,p=0.017,说明hla-drb1*09不仅是丙肝感染的抵抗基因,还在阻碍alb水平下降及疾病进展的过程中起到保护作用。12、hla-a位点基因频率(高分辨)与抗体亲和力呈正相关,提示基因频率越高,抗体亲和力越强。13、对携带hla-a*02,hla-b*39,hla-drb1*09,hla-drb1*11,hla-a*33,hla-b*58,hla-drb1*07,hla-drb1*13基因型别的患者进行抗体亲和力、抗体物质量以及抗体总活性的比较,由于hla-b*39仅有1例阳性患者,hla-drb1*11只有4例阳性患者,故不对此两种基因型别进行比较。结果显示,按抗体亲和力排序,hla-a*33hla-b*58hla-drb1*07hla-a*02hla-drb1*09hla-drb1*13。14、生物信息学角度的亲和力分析结果是:hla对hcvns3的亲和性排序为:hla-b*58hla-a*33hla-drb1*09hla-drb1*07hla-a*02hla-drb1*13。结论:1.hcv抗体多维数据解析方法成立,hcv感染患者抗体亲和力、抗体总活性、抗体物质量的变化有可能反应肝细胞损伤及病毒载量的变化,具有一定的临床应用价值。2.hcv抗体多维数据解析发现,抗体亲和力与病情联系密切,优于传统实验室仅检测抗体总活性的方法。抗体亲和力在疾病转归中可能有一定的指示作用,具有较高的诊断效能,高亲和力抗体可能有利于疾病的好转,测定抗体亲和力将有助于临床医生判断疾病的预后。3.HLA多态性与丙型肝炎发生发展过程及HCV抗体亲和力高低有关,基因频率高(进化保守),可能越有利于产生高亲和力的抗体。
[Abstract]:Background: Hepatitis C Virus (HCV) is the main pathogen of post transfusion and sporadic hepatitis and is a worldwide public health problem. Around the world, about 170 million HCV serologically positive patients face the risk of transforming into liver cirrhosis and liver cancer. Although HCV can stimulate the body to produce an effective immune response, but a 80% HCV infection In patients with chronic hepatitis C, only 20% of the patients can effectively remove the virus, recover spontaneously and without any sequelae. Most of the patients with HCV chronic infection can produce antibodies against different HCV virus proteins and detect the presence of these antibodies in the patient's serum. Obviously, the type of host immune response to HCV infection and the type of host immune response are clearly defined. Strength may play an important role in its prognosis for its prognosis,.HCV antibody detection is of great value for the diagnosis of HCV. Neutralizing antibodies can directly act on HCV virus, but it is generally believed that the antibodies commonly detected in HCV infected persons are directed against the HCr43 protein (HCV unstructured region NS3 and core region), c100-3 antigen (HCV non structural NS3, NS4 coding). The antibody, as a non neutralizing antibody, can not determine the immune state of the HCV infected person by detecting the HCV antibody. We found the same non neutralizing antibody in the association of anti-HBc affinity with HBV infection, but the affinity of anti-HBc is closely related to the prognosis of HBV infection; and there are also studies showing that the affinity of antibodies is high and low. It is possible to accurately reflect the activity of cytomegalovirus infection. The determination of HCV antibody affinity may be more accurate to reflect the immune status of HCV infected people and provide valuable information for determining the status and outcome of HCV infection. The total activity of antibodies depends on the content of antibody protein and its binding activity with the anti primitive (affinity). The test of HCV antibody in bed laboratory only detected the total activity of antibody, and did not involve the detection of antibody protein content and antibody affinity. The total activity of HCV antibody was analyzed into antibody protein and antibody affinity, and the detection system of the general activity of HCV antibody was tried to refer to the detection principle of the affinity of hepatitis B anti-HBc. To establish a method to detect the affinity of HCV antibody, and to explore the relationship between the antibody affinity of HCV, the total activity and the quality of the virus, the degree of body damage and the dynamics of the changes in different stages of the disease. The study on the persistence and clearance of HLA and HCV infection in the last ten years is not much, and the immune response of the body to the epitopes of the virus depends on the immune response of the body. Antigen presenting cells are capable of processing and presenting antigen epitopes, and the ability of immune cells to identify these epitopes, which are genetically determined by HLA molecules. Therefore, it is speculated that the HLA type can affect the.Hla-i allele of HCV infection and the self limiting transformation of HCV infection, such as the HLA-I class, etc. The gene A3, B27 and cw*01 are all associated with virus clearance, while B8 is highly compatible to HLA-DR in the.Ns3 region associated with the persistent infection of the virus, suggesting that the hla- class II molecules are also related to the virus clearance. Some hla- class II alleles are also related to the outcome of HCV infection, and the most highly related hla- II class of the virus clearance. The allele is dqb1*0301 gene and drb1*1101., however, the results of these studies are not entirely consistent and even contradictory. Objective: to establish a detection method for affinity of HCV antibodies with antibody multidimensional data analysis, and to explore the changes in the affinity, mass and total activity of HCV antibody, the amount of the virus and the damage process of the body. The relationship between.Hcv and HLA-A, HLA-B, HLA-DRB1 genotypes and the process of disease and its mechanism of action. Methods: 1, the three subcutaneous injection of Recombinant Hepatitis B Vaccine was used to immunize rabbits repeatedly, and the rabbit blood was collected for the first week after the first injection, and the low affinity antibody animal serum was obtained; the third injection was given. After two weeks, rabbit serum was collected to obtain high affinity antibody animal serum, double dilution sera, and double antigen sandwich enzyme-linked immunosorbent assay was used to detect anti-hbs. in 106 cases of HCV infected patients, and HCV antibody was determined by chemiluminescent particle immunization assay, and HCV-RNA was detected by pcr- fluorescence probe. Detection of alt, AST, ALB and other clinical indicators. According to alt, AST level and HCV-RNA level in three groups, the affinity of antibody, the total activity of antibody and the change of the quality of antibody were compared and analyzed, and 328 cases of HCV infected patients were randomly collected, and the HCV antibody and pcr- fluorescence were measured by the chemiluminescent particle immunoassay. HCV-RNA was detected by probe, and alb. was divided into chronic hepatitis group and liver cirrhosis group according to the disease stage. The chronic hepatitis group was divided into ALB normal HCV-RNA negative group (improvement recovery stage) and other group (progressive stage) according to the level of ALB and HCV-RNA, and the changes of antibody affinity, antibody quality and total antibody activity were analyzed and compared. 3, 104 patients with HCV infection and 200 healthy controls were randomly collected. HCV antibody was measured by chemiluminescent particle immunoassay, HCV-RNA was detected by pcr- fluorescence probe, and serum ALB level was detected. The identification of HLA genotypes was carried out by sequencebasedtyping (PCR-SBT). The different HLA types were compared and analyzed. ALB level, antibody affinity, antibody quality and antibody total activity change.4, using online search database combined with discoverystudio (DS) software to analyze and predict the interaction between the 6 kinds of HLA protein fractions of NS3 and hla-drb1*09, hla-a*02, hla-a*33, hla-b*58, hla-drb1*07, hla-drb1*13. The database includes national national medical books Library literature retrieval system NCBI, protein structure database PDB and PROSITE database. Results: 1, the serum antibody affinity of female rabbits after first times immunization is 0.522, second times 0.826 and third times 0.896. The affinity of serum antibody after first immunization of male rabbits is 0.816, second is 0.846 and third is 0.972, which are all gradually increasing and affinity with antibody. The antibody quality of.2, alt, AST normal group was significantly lower than any abnormal group (P0.05), and the antibody affinity was significantly higher than any abnormal group (P0.05). The total antibody activity in the three groups was not significantly different (P0.05).3. According to HCV-RNA level, the normal group of ALT and AST (53 cases) were further divided into two groups. In the group of HCV-RNA negative group and HCV-RNA positive group, the total antibody activity of.Hcv-rna negative group was significantly lower than that of HCV-RNA positive group (P0.05), but the antibody affinity was significantly higher than that of HCV-RNA positive group (P0.05).4, the total antibody activity of the HCV-RNA negative group was significantly lower than the low viral load, and the high virus load group (P0.05), The antibody affinity was significantly higher than the low viral load, high viral load group (P0.05).5, HCV-RNA positive as the dependent variable, antibody affinity, antibody quality and antibody total activity as independent variables, logistic regression analysis, HCV-RNA level with antibody affinity was negatively correlated (P0.05).6, ALB normal HCV-RNA negative group antibody affinity obviously Higher than the other groups of chronic hepatitis and liver cirrhosis (P0.05), the quality of antibody was significantly lower than that of other groups of chronic hepatitis and liver cirrhosis (P0.05). The total antibody activity was not significantly different in the three groups (P0.05).7, and the area of antibody affinity and antibody mass was 0.816,0.811, respectively, which showed high diagnostic efficiency. It was significantly higher than the traditional antibody test (the area under the ROC curve was 0.581). The antibody affinity was 0.873 for the best diagnostic critical point for differentiating the progression of hepatitis C infection (HCV-RNA positive) and the good turn phase (HCV-RNA negative). The higher the antibody affinity, the less the virus replication was.8, and the ratio of the high antibody affinity (> 0.873) in the alb normal HCV-RNA negative group. The cases were significantly higher than the other groups of chronic hepatitis and liver cirrhosis (P0.05).9, the serum of HCV-RNA (+) was neutralized with high affinity antibody, and the lgrna was significantly lower than that before the neutralization level (P0.05).10. In HCV patients and the healthy control group, the 8 HLA types were identified with significant difference (P0.05), hla-a*02, hla-b*39, hla-drb1*09, respectively. Drb1*11, hla-a*33, hla-b*58, hla-drb1*07, hla-drb1*13., hla-a*02, hla-b*39, hla-drb1*09, hla-drb1*11 in HCV patients were significantly lower than those in the healthy control group. Anti gene, while hla-a*33, hla-b*58, hla-drb1*07, and hla-drb1*13 are the sensitive gene.11 of hepatitis C infection. In these 8 HLA types, serum ALB levels are in hla-a*02, hla-a*33, hla-b*39, hla-drb1*07, hla-drb1*11, carriers and non carriers. The level of Alb is obviously different, the former p=0.015 and the latter p=0.003, all 0.05, but only hla-drb1*09 in cross validation still have significant differences, p=0.017, indicating that hla-drb1*09 is not only the resistance gene of hepatitis C infection, but also hinders the decline of ALB and the progression of the disease, which plays a protective role.12, the frequency of the HLA-A site gene (high resolution). There was a positive correlation with antibody affinity, suggesting that the higher the frequency of the gene, the stronger the antibody affinity.13, the antibody affinity, the quality of the antibody and the total activity of the antibody in patients carrying hla-a*02, hla-b*39, hla-drb1*09, hla-drb1*11, hla-a*33, hla-b*58, hla-drb1*07, and hla-drb1*13, because hla-b*39 only had 1 positive patients. Hla-drb1*11 only 4 positive patients did not compare the two genotypes. The results showed that according to the affinity sequencing of antibody, the affinity analysis of hla-a*33hla-b*58hla-drb1*07hla-a*02hla-drb1*09hla-drb1*13.14 and bioinformatics was: the affinity of HLA to hcvns3 was: hla-b*58hla-a*33hla-drb1*09hla-drb1*07hl A-a*02hla-drb1*13. conclusion: the multi-dimensional data analysis method of 1.hcv antibody was established. The antibody affinity, the total activity of the antibody and the change of the quality of the antibody may reflect the changes of the liver cell damage and the viral load. It has certain clinical application value of the.2.hcv antibody multidimensional data analysis, and the affinity of the antibody is closely related to the condition of the disease. It is better than the traditional laboratory to detect the total activity of antibody only. The antibody affinity may have a certain indicator in the outcome of the disease. It has a high diagnostic efficiency. The high affinity antibody may be beneficial to the improvement of the disease. The determination of antibody affinity will help clinicians to judge the prognosis of the.3.HLA polymorphism and the incidence of hepatitis C The development process is related to the high affinity of HCV antibody. The high gene frequency (evolutionary conservatism) may help to produce high affinity antibodies.
【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.63;R446.6

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