抗病毒治疗乙肝病人TCR可变区的深度测序分析

发布时间：2018-07-09 17:16

本文选题：慢性乙型肝炎 + 细胞毒性T细胞　；参考：《河南大学》2014年硕士论文

【摘要】：背景: 据世界卫生组织报道，全球约有20亿人曾感染过HBV，并且3.5亿人为慢性感染者，每年大约有100万人死于HBV感染所导致肝硬化、肝衰竭和原发性肝癌。我国为慢乙肝流行高发区域，慢性HBV感染者约9300万，其中有症状需要治疗的活动性乙型肝炎患者约为2000多万。慢性乙型肝炎患者的抗病毒疗效受病毒自身和宿主等因素的影响，而宿主的细胞免疫状态是治疗应答的重要因素之一，在肝细胞的损害及病毒的清除作用中发挥重要的作用。在急性自限性HBV感染中，，针对HBV的细胞免疫反应比较强，然而在慢乙肝中，针对HBV的细胞免疫反应要弱得多。感染的病毒通过免疫介到引起肝细胞的损害，T细胞功能异常可能是导致HBV感染慢性化的根本原因。目前的慢乙肝主要治疗措施是：保肝药物、抗病毒及免疫调节治疗，均不能彻底根除乙肝。在HBV的免疫应答过程中，特异性细胞毒性T细胞（CTL）决定了疾病的转归及在清除病毒中发挥了重要作用。T细胞受体（T cell receptor,TCR）决定了CTL特异性，TCR的特性主要取决于可变区V区的分子组成。研究TCR的组成特点有助于深入理解HBV免疫应答的反应机制。近年来，随着新一代测序技术的飞速发展和人类基因组计划的完成，高通量测序技术（High-throughput sequencing）即深度测序和“下一代”测序（Next genenrationsequencing）具有高通量、操作平台简易、质量高等优点，已被应用医学的各个领域。本实验主要利用慢性乙型肝炎患者治疗前后的外周血淋巴细胞扩增β链可变受体区域进行高通量测序，分析受体区CDR的种类变化及克隆株的变化，从而帮助阐明CHB患者的细胞免疫应答的特点机制，为揭示慢性乙型肝炎的发病机制、有效治疗策略提供实验研究的新思路及技术平台，从而为慢性乙型肝炎的免疫治疗提供帮助。目的：利用Ion Torrent高通量测序技术观察慢乙肝患者治疗前后TCR β链V区家族性基因的差异性表达，为深入探讨慢乙肝患者的发病机制奠定实验基础。材料与方法：（1）研究对象：收集2010年3月-2013年12月就诊于首都医科大学附属北京佑安医院门诊的临床治疗有效的HBeAg阳性的慢性乙型肝炎患者，所有患者均符合2010年版《慢性乙型肝炎防治指南》的诊断标准。排除合并其他病毒感染、其他原因引起的肝病、慢乙肝不适合抗病毒治疗的患者。（2）方法：收集慢乙肝患者的外周血，分离单个核细胞；提取总RNA后提取mRNA；逆转录合成cDNA,后扩增TCRβ链V区基因，通过切胶回收后制备文库高通量测序。结果：样品产生的380,000reads,同时考虑V、J家族基因，将所有30个V家族和J家族的基因构建数据库，做Blast，发现有15个基因上调，2个基因(TRBV28_TRBJ1.5，TRBV6_TRBJ2.10)上调最为明显，有统计学意义。结论1.通过高通量测序，挑选出了TRBV/TRBJ在慢乙肝患者CD8+T细胞的异常表达，TRBV10_TRBJ2.1,TRBV10_TRBJ2.2,TRBV10_TRBJ2.7,TRBV12_TRBJ1.1,TRBV27_TRBJ2.2TRBV28_TRBJ1.5，TRBV6_TRBJ2.1,TRBV6_TRBJ2.1可能与HBV的发生及发展相关。 2.通过比较分析，初步推测TRBV28_TRBJ1.5，TRBV6_TRBJ2.1可能与HBV治疗效果相关。
[Abstract]:Background:
According to the WHO, about 2 billion people in the world have been infected with HBV, and 350 million people with chronic infection, about 1 million people die each year from HBV infection, liver cirrhosis, liver failure and primary liver cancer.
China is a high incidence region of chronic hepatitis B epidemic, with about 93 million chronic HBV infection, of which about 20000000 of the patients with active hepatitis B are treated with symptomatic treatment. The antiviral effect of chronic hepatitis B patients is affected by the factors such as the virus itself and host, and the host cell immune state is one of the important factors for the treatment response. Cell damage and virus scavenging play an important role. In acute self limiting HBV infection, the cellular immune response to HBV is stronger. However, in the slow hepatitis B, the cellular immune response to HBV is much weaker. The infected virus can cause liver cell damage through immunization, and the abnormal function of T cells may lead to the sense of HBV. The main treatment of chronic hepatitis B is that the main treatment measures of chronic hepatitis B are: liver preservation drugs, antiviral and immunomodulatory treatment, all can not eradicate hepatitis B thoroughly. In the process of HBV's immune response, specific cytotoxic T cells (CTL) determine the outcome of the disease and play an important role in the clearance of the disease (T cell Recep). Tor, TCR) determines the specificity of CTL. The characteristics of TCR mainly depend on the molecular composition of the V region of the variable region. The study of the composition of TCR helps to understand the reaction mechanism of the HBV immune response in depth.
In recent years, with the rapid development of new generation sequencing technology and the completion of human genome project, the high throughput sequencing technology (High-throughput sequencing), that is, the depth sequencing and the "next generation" sequencing (Next genenrationsequencing) have high throughput, simple operating platform and high quality, and have been applied in various fields of medical science. We mainly use the peripheral blood lymphocytes of patients with chronic hepatitis B to amplify beta chain variable receptor region to carry out high flux sequencing, analyze the variety of CDR and the change of clones, so as to help clarify the characteristic mechanism of CHB patients' cellular immune response, and to uncover the pathogenesis of chronic hepatitis B and effective treatment. The strategy provides new ideas and technology platform for experimental research, thus providing help for immunotherapy of chronic hepatitis B.
Objective:
The differential expression of family genes in TCR beta chain V region before and after treatment of chronic hepatitis B patients was observed by high throughput sequencing of Ion Torrent, which lay an experimental basis for exploring the pathogenesis of chronic hepatitis B patients.
Materials and methods:
(1) the subjects were to collect the clinical treatment of HBeAg positive chronic hepatitis B patients in the outpatient clinic of Beijing, Beijing, March 2010. All patients were in accordance with the 2010 edition of the guidelines for the prevention and treatment of chronic hepatitis B. Liver disease, slow hepatitis B is not suitable for patients with antiviral treatment.
(2) methods: the peripheral blood of patients with chronic hepatitis B was collected, mononuclear cells were isolated, mRNA was extracted after total RNA extraction, cDNA was synthesized by reverse transcriptase, and then TCR beta chain V region gene was amplified, and high throughput sequencing of the library was prepared by cutting glue.
Results: the 380000reads produced by the sample, taking into account the V, J family genes, constructed the database of all 30 V families and the J family genes, made Blast, found that 15 genes were up regulated, and 2 genes (TRBV28_TRBJ1.5, TRBV6_TRBJ2.10) were up - regulated, with statistical significance.
Conclusion 1. through high throughput sequencing, the abnormal expression of TRBV/TRBJ in CD8+T cells in patients with chronic hepatitis B was selected. TRBV10_TRBJ2.1, TRBV10_TRBJ2.2, TRBV10_TRBJ2.7, TRBV12_TRBJ1.1, TRBV27_TRBJ2.2TRBV28_TRBJ1.5, TRBV6_TRBJ2.1, TRBV6_TRBJ2.1 may be related to the occurrence and development of HBV.
2. through comparative analysis, it is preliminarily speculated that TRBV28_TRBJ1.5 and TRBV6_TRBJ2.1 may be related to the therapeutic effect of HBV.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.62

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