本文選題：慢性乙型肝炎 + 細胞毒性T細胞�。� 參考：《河南大學》2014年碩士論文

【摘要】：背景: 據(jù)世界衛(wèi)生組織報道，全球約有20億人曾感染過HBV，并且3.5億人為慢性感染者，每年大約有100萬人死于HBV感染所導致肝硬化、肝衰竭和原發(fā)性肝癌。 我國為慢乙肝流行高發(fā)區(qū)域，慢性HBV感染者約9300萬，其中有癥狀需要治療的活動性乙型肝炎患者約為2000多萬。慢性乙型肝炎患者的抗病毒療效受病毒自身和宿主等因素的影響，而宿主的細胞免疫狀態(tài)是治療應答的重要因素之一，在肝細胞的損害及病毒的清除作用中發(fā)揮重要的作用。在急性自限性HBV感染中，，針對HBV的細胞免疫反應比較強，然而在慢乙肝中，針對HBV的細胞免疫反應要弱得多。感染的病毒通過免疫介到引起肝細胞的損害，T細胞功能異�？赡苁菍е翲BV感染慢性化的根本原因。目前的慢乙肝主要治療措施是：保肝藥物、抗病毒及免疫調節(jié)治療，均不能徹底根除乙肝。在HBV的免疫應答過程中，特異性細胞毒性T細胞（CTL）決定了疾病的轉歸及在清除病毒中發(fā)揮了重要作用。T細胞受體（T cell receptor,TCR）決定了CTL特異性，TCR的特性主要取決于可變區(qū)V區(qū)的分子組成。研究TCR的組成特點有助于深入理解HBV免疫應答的反應機制。 近年來，隨著新一代測序技術的飛速發(fā)展和人類基因組計劃的完成，高通量測序技術（High-throughput sequencing）即深度測序和“下一代”測序（Next genenrationsequencing）具有高通量、操作平臺簡易、質量高等優(yōu)點，已被應用醫(yī)學的各個領域。本實驗主要利用慢性乙型肝炎患者治療前后的外周血淋巴細胞擴增β鏈可變受體區(qū)域進行高通量測序，分析受體區(qū)CDR的種類變化及克隆株的變化，從而幫助闡明CHB患者的細胞免疫應答的特點機制，為揭示慢性乙型肝炎的發(fā)病機制、有效治療策略提供實驗研究的新思路及技術平臺，從而為慢性乙型肝炎的免疫治療提供幫助。 目的： 利用Ion Torrent高通量測序技術觀察慢乙肝患者治療前后TCR β鏈V區(qū)家族性基因的差異性表達，為深入探討慢乙肝患者的發(fā)病機制奠定實驗基礎。 材料與方法： （1）研究對象：收集2010年3月-2013年12月就診于首都醫(yī)科大學附屬北京佑安醫(yī)院門診的臨床治療有效的HBeAg陽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》的診斷標準。排除合并其他病毒感染、其他原因引起的肝病、慢乙肝不適合抗病毒治療的患者。 （2）方法：收集慢乙肝患者的外周血，分離單個核細胞；提取總RNA后提取mRNA；逆轉錄合成cDNA,后擴增TCRβ鏈V區(qū)基因，通過切膠回收后制備文庫高通量測序。 結果：樣品產(chǎn)生的380,000reads,同時考慮V、J家族基因，將所有30個V家族和J家族的基因構建數(shù)據(jù)庫，做Blast，發(fā)現(xiàn)有15個基因上調，2個基因(TRBV28_TRBJ1.5，TRBV6_TRBJ2.10)上調最為明顯，有統(tǒng)計學意義。 結論1.通過高通量測序，挑選出了TRBV/TRBJ在慢乙肝患者CD8+T細胞的異常表達，TRBV10_TRBJ2.1,TRBV10_TRBJ2.2,TRBV10_TRBJ2.7,TRBV12_TRBJ1.1,TRBV27_TRBJ2.2TRBV28_TRBJ1.5，TRBV6_TRBJ2.1,TRBV6_TRBJ2.1可能與HBV的發(fā)生及發(fā)展相關。 2.通過比較分析，初步推測TRBV28_TRBJ1.5，TRBV6_TRBJ2.1可能與HBV治療效果相關。
[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.
【學位授予單位】：河南大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.62

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