摘要
研究背景：在全世界范围内大约有3亿5千万人为慢性HBV感染者。HBV感染可造成慢性乙型肝炎、肝硬化、肝癌，每年大约有一百万人死于HBV感染。HBV的复制并没有直接的肝细胞毒性，在无症状乙肝病毒携带者体内尽管HBV长期持续复制，但是引起的肝脏损伤却很轻微。真正引起肝脏损伤的是针对肝细胞表面的病毒抗原引起的宿主免疫应答，即细胞免疫反应是导致肝细胞损伤的直接原因，那些存在免疫功能缺陷的患者在感染了HBV后，只有很轻的肝脏损伤，大部分患者会发展为慢性携带状态。针对HBV的免疫反应以及这些免疫反应在乙型肝炎的发病机制中的作用并不完全明了。相关的临床研究表明，在急性自限性乙型肝炎患者的外周血中，可检测到针对许多HBV抗原的强烈的T细胞反应。相比较，在慢性HBV携带者，病毒特异性的T细胞反应大大地减弱。这些都表明T细胞免疫反应，特别是细胞毒性T细胞反应对于病毒的清除起着非常重要的作用。T细胞受体（TCR)是存在于T细胞表面的特异性抗原识别受体，TCR识别抗原决定簇后能活化T细胞，产生一系列的免疫应答。T细胞所具有的高度特异性抗原识别和产生免疫效应特性依赖于具有高度多样性的TCR。HBV感染后,根据HBV感染的自然进程可形成4个不同的阶段：免疫耐受期、免疫清除期、非活动性携带期和再活动期。针对无症状HBV携带者和慢性活动性乙型肝炎患者体内TCR研究，有利于了解HBV慢性感染者免疫耐受机制和细胞免疫功能，有利于采用适当合理的治疗手段。
目的:了解无症状HBV携带者外周血CD4+和CD8+TCR Vβ基因家族克隆化特征，了解它们在无症状HBV携带者体内的免疫耐受状态中所起的作用。通过对慢性活动性乙型肝炎患者外周血CD4+和CD8+TCR Vβ基因家族克隆化特征的研究，了解它们在免疫应答中所发挥的作用；对TCR Vβ家族中出现单克隆扩增的家族的CDR3进行基因测序分析，比较其氨基酸序列，了解有无共同的序列，寻找在免疫应答中的共同抗原表位；对无症状HBV携带者和慢性活动性乙型肝炎患者PBMCs经HBsAg刺激后，分泌的IL-4和IFN-γ进行检测，探讨Th1/Th2型细胞因子在HBV慢性感染中所起的作用。
方法:采用RT-PCR-基因扫描-序列分析法对CD4+/CD8+TCR Vβ家族克隆化特征检测。选取12例无症状HBV携带者和10例慢性活动性乙型肝炎患者，采抗凝血10-20ml，分离外周血单个核细胞（PBMCs)，免疫磁珠分离CD4+和CD8+T细胞，提取总RNA，逆转录合成cDNA。巢式PCR扩增为两步法，针对TCR Vβ24个基因家族设计两轮引物，扩增24个基因家族。然后将PCR产物用373DNA自动测序仪上电泳，并用扫描软件对结果进行分析和定量，并绘制出扫描图，分析哪些家族是单克隆或寡克隆扩增。对单克隆扩增的TCR VβCDR3进行测序，结果用DNAMAN software version1.0软件进行分析，并将核苷酸序列，氨基酸序列进行比较。了解有无共同的氨基酸序列。采用ELISA方法对无症状HBV携带者和慢性活动性乙型肝炎患者PBMCs经特异性抗原刺激后，培养上清液中的的IL-4和IFN-γ水平进行检测。
结果:全部健康对照者的CD4+和CD8+TCR Vβ各个家族的基因扫描图均可见中间高两边低的钟形图，为高斯分布。在所有的12例无症状HBV携带者外周血CD4+或CD8+TCR Vβ家族中，均可检测到单克隆或寡克隆扩增的家族。其中CD4+TCR Vβ家族中出现克隆化扩增的家族为Vβ2、3、4、5、7、8、9、11、12、15、18和21，其中Vβ7、9、12、15家族出现的频率高于其它家族。而CD8+TCR Vβ家族，12个患者中只有6个患者的TCR Vβ家族出现克隆化扩增，出现在Vβ3、5、7、9、11、12、16、18和21家族中。并且CD4+TCR Vβ家族中出现克隆化扩增的家族数量（n=24)明显高于CD8+TCR Vβ家族（n=9）（P<0.05)。基因测序结果显示，这些单克隆扩增家族CDR3并未发现共同氨基酸序列。10例慢性乙肝患者CD4+和CD8+TCRVβ分别在不同的家族出现单峰和寡峰，CD4+和CD8+TCR Vβ家族出现克隆性扩增的家族的数量没有明显差异。CD4+TCR Vβ家族出现14个克隆化扩增的家族，出现在Vβ2、5、7、9、11、12、13、15这些家族中，其中Vβ2、9、11家族出现克隆化扩增的频率较其他家族高。CD8+TCR Vβ家族出现18个克隆化家族，出现在Vβ2、6、7、8、9、10、11、12、21、22这些家族，其中Vβ7、8、9、11、12家族出现克隆化的频率较其他家族高。部分CD8+TCR Vβ单克隆扩增的家族之间存在部分相同的基序，其中患者2的Vβ12家族、患者8的Vβ12家族、患者9的Vβ7家族有共同氨基酸序列“PR”,患者3的Vβ8家族、患者5的Vβ11家族、患者9的Vβ7和Vβ11家族、患者10的Vβ12家族有共同氨基酸序列“TL”。CD4+和CD8+TCRVβ家族中，出现克隆化扩增的家族的数量并没有明显差别。对两种患者及健康对照者PBMCs经HBsAg刺激后培养，检测培养液上清中的IL-4和IFN-γ水平的结果为，无症状HBV携带者PBMC经特异性抗原刺激后，IL-4水平明显高于对照组，IFN-γ水平明显低于对照组；对慢性活动性乙型肝炎患者，其PBMCs分泌的IFN-γ水平明显高于对照组，而IL-4水平与对照组没有明显差异。
结论:1.无症状HBV携带者体内存在不同CD4+和CD8+TCR Vβ家族的克隆性扩增,并且是以CD4+TCR Vβ家族扩增为主。 CD4+和CD8+单克隆扩增的TCR Vβ家族的CDR3经基因测序后均未发现相同的氨基酸序列，这说明HBV抗原表位存在着多样性，多个抗原表位可刺激T细胞发生免疫应答。2.慢性活动性乙型肝炎患者存在不同的CD4+和CD8+TCR Vβ家族克隆性扩增。 CD8+TCR Vβ部分单克隆扩增的家族的CDR3经基因测序后，发现存在共同基序“PR”和“TL”，这提示不同的TCR Vβ家族可以识别不同抗原表位的相同部分。3.Th1/Th2细胞因子的分泌失衡可能是引起HBV持续性感染的重要原因。
我们采用免疫磁珠法将PMBC分离为CD4+和CD8+T细胞两个亚群，分别对两个细胞群的TCR Vβ基因家族克隆化特征进行分析，明确在无症状HBV携带者和慢性活动性乙型肝炎患者体内，导致免疫耐受或免疫应答的是哪一个细胞亚群。利用RT-PCR-基因扫描-序列分析的方法确定在HBV刺激后TCR基因重排的克隆性，TCR基因的CDR3长度和序列，就有可能确定抗原特异性TCR克隆。通过分析病毒抗原相关的TCR基因，对开展相应的抗病毒特异性免疫治疗是非常有意义的。
Background：Hepatitis B virus (HBV) infects more than350million people worldwide.Hepatitis B is a leading cause of chronichepatitis,cirrhosis and hepatocellular carcinoma,accounting for1million deaths annually. The HBV replication cycle is not directlycytotoxic to cells. The fact accords well with the observation thatmany HBV carriers are asymptomatic and have minimal liverinjury,despite extensive and ongoing intrahepatic replication of thevirus. It is now thought that host immune responses to viral antigensdisplayed on infected hepatocytes are the principal determinantsof hepatocellular injury. This notion is consistent with the clinicalobservation that patients with immune defects who are infected withHBV often have mild acute liver injury but high rates of chroniccarriage. The immune responses to HBV and their role in thepathogenesis of hepatitis B are not completed understood.Correlative clinical studies show that in acute, self-limitedhepatitis B, strong T-cell responses to many HBV antigens are readilydemonstrablein the peripheral blood.By contrast,in chronic carriersof HBV, such virus-specific T-cellresponses are greatly attenuated, at least as assayed in cells from the peripheral blood.This patternstrongly suggests that T-cell responses, especiallythe responses ofcytotoxic T lymphocytes,play a central role in viral clearance. Tcell antigen receptor(TCR) recognize antigen epitope specifically,which can result in immune response.Those patientswith chronic HBVinfection may present in one of the four phases of infection: immunetolerance, immuneclearance, inactive carrier state,andreactivation. We study the TCR clonity of chronic asymptomaticcarriers and chronic hepatitis B to understand the status of cellimmune responses,which can guide to reasonable treatment of HBVpersistent infection.
Objectives: To understand the clonity charateristic of CD4+and CD8+TCR in chronic asymptomatic carriers and to study the role of cellimmune response in the state of immune tolerance; To study the clonitycharateristic of CD4+and CD8+TCR in chronic hepatitis B and toinvestigate the role of cell immune response in the state of immuneclearance; To study if common nuleotide sequence of monoclonalTCR VβCDR3by nucleotide sequencing and comparison and to look forthe common HBV epitope in cell immune response. To clarify the roleof Th1/Th2cytokines in chronic HBV infection through detecting IL-4and IFN-γ level in supernatant of cultured PBMCs.
Methods: Chronic asptomatic carriers and patients with chronic hepatitis B were enrolled in the study. RT-PCR-genescan-sequencewas used in our study.10-20ml heparinized blood samples werecollected from every patients. CD4+and CD8+T lymphocytes wereisolated using monoclonal antibody-coated magnetic beads and thenRNA was extracted. cDNA was synthesized using RNA, reversetranscriptase and oligo dT in a total volume of20ul. Using a two-stepPCR assay, CDR3length within the TCR Vβ chain was analyzed. ForwardV β and reverse C β primers of two rounds were designed toamplified the24TCR Vβ families. PCR products were analyzed usingaApplied Biosystems model373DNA sequencer. The data were analyzedand quantified using the ABIPRISM GeneScan analysis software todetermine the oligoclonal or monoclonal families.The amino acidsequence of the CDR3in monoclonal Vβ families were analyzedusing DNAMAN software version1.0. ELISA was used to detect IL-4and IFN-γ level in the supernatant of cultured PBMCs which werestimulated by HBsAg.
Results: In this study, the results of genescan analysis showedthat the CD4+and CD8+TCR Vβfamilies of the healthy control donorsshowed Gaussian distribution.However, some CD4+and CD8+TCR Vβ families showed monoclnal and oligoclonal expansion in allthe12chronic asymptomatic HBV carriers. TCR Vβ2、3、4、5、7、8、9、11、12、15、18and21families showed clonal expansion. The expansion frequencies of TCRVβ7、9、12and15families were higherthan the others TCR Vβfamilies.In CD8+TCR Vβ families,6among12patients showed clonal expansion and the TCR Vβfamilies were Vβ3、5、7、9、11、12、16、18and21.The clonal expansion number ofCD4+TCR Vβ families (n=24) was significantly higer than that ofCD8+TCR Vβ families(n=9). The sequence results showed that nocommon amino acid exist in the monoclonal TCR Vβ CDR3. Clonalexpansion of CD4+and CD8+TCR Vβfamilies can also be seen in10patients with chronic hepatitis B. In CD4+TCR Vβfamilies,Vβ2、5、7、9、11、12、13、15families showed clonal expasion and theexpansion frequencies of Vβ2、9、11families were higher than theothers families. In CD8+TCR Vβfamilies,Vβ2、6、7、8、9、10、11、12、21、22showed clonal expasion and the expansion frequenciesof Vβ7、8、9、11、12were higher than the others families. Therewas no significant difference in cloanl expansion number betweenCD4+and CD8+TCR Vβfamilies. The sequence results showed that someCD8+TCR Vβ families shared the same motif. TCR Vβ12families ofcase2and case8, Vβ7family of case9shared the same motif “TL”and TCR Vβ8family of case3, TCR Vβ11family of case5, TCR Vβ7、11families of case9shared the same motif “TL”.The resultsof IL-4and IFN-γ levels in the supernatant of cultered PBMC showedthat IL-4level in asymptomatic HBV carriers was signigicantly higer than that in healthy controls and that IFN-γ level inasymptomatic HBV carriers was signigicantly lower than that inhealthy controls.In chronic active hepatitis B, the IFN-γ level wassignificantly higher than that in healthy controls and there was nosignificantly difference in IL-4level between the two groups.Conclusions: In chronic asymptomatic HBV carriers, the monoclonalexpansion of CD4+TCR Vβfamilies play an important role in cellularimmune tolerance. In addition, different TCR CDR3sequences inmonoclonal expansion families indicated that many HBV antigens wereinvolved in the celluar immune response. In patient with chronicactive hepatitis B, both CD4+and CD8+T cell are crucial incellular immune response. Common amino acid motif “LF” and “PR”in some monoclonal CD8+TCR VβCDR3suggested that they may recognizethe same regions of different antigen epitopes; Loss of Th1/Th2cytokines balance plays an important role in immunopathogenesis ofchronic HBV infection.
The previous study in TCR Vβclonity of patients with HBV infectionwas focused on PBMCs,not CD4+and CD8+Tsubsets. The results maybe disurbed each other. In our study, CD4+and CD8+T subsets wereisolated using antibodies coated magnetic beads, and then the clonitycharacteristics of CD4+and CD8+TCR Vβfamilies were analyzedrespectively to investigate the role of the two subsets in the pathogenesis of chronic HBV infection.RT-PCR-Genescan-sequence wasused to analyze the TCR clonity，the length and the sequence ofTCR CDR3of patients with chronic HBV infection, which may helpdetermine the antigen-specific TCR clone. It is useful to guide tothe reasonable treatment of HBV persistent infection.
引文
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