强直性脊柱炎患者外周血T细胞受体β链可变区基因谱系多态性的初步研究

目的 探讨强直性脊柱炎(AS)患者外周血T细胞受体β链可变区互补决定区3(TCRBV CDR3)谱系多态性,为AS免疫发病机制研究提供实验依据.方法 采用反转录-聚合酶链反应(RT-PCR)扩增AS患者外周血单个核细胞(PBMC)中T细胞TCR BV的26个亚家族CDR3,经免疫扫描谱型技术分析TCR BV CDR,3的谱系多态性情况.结果 20例活动期AS患者TCR BV CDR3扫描谱型均出现非正态的异常峰型,包括单峰、寡峰/寡峰趋势、偏峰和不规则异常峰型.其中有18例患者部分亚家族出现寡克隆/寡克隆趋势增生,有1例患者BV16和BV18的2个亚家族出现单克隆增生.5名健康对照PBMC TCR BV CDR3谱型绝大多数呈高斯分布.结论 AS患者外周血TCR BV CDR3谱系具有显著多态性和谱系漂移特点,进一步表明T细胞在AS免疫发病机制中扮演重要角色.单/寡克隆增生的T细胞有可能是AS发病中的自身反应性T细胞.     

慢性乙型肝炎患者外周血CD8~+T淋巴细胞亚群T淋巴细胞受体β链互补决定区3谱型研究

目的 研究慢性乙型肝炎(CHB)患者外周血CD8+T淋巴细胞亚群T淋巴细胞受体(TCR)β链Ⅴ区(BV)中互补决定区3(CDR3)谱型特点.方法 采集8例CHB患者(ALT>2倍正常值上限)的抗凝血,分离外周血单个核细胞,磁珠分选CD8~+T淋巴细胞亚群,提取RNA并应用逆转录-聚合酶链反应扩增TCR BV中CDR3基因的24个家族,采用免疫指纹技术进行TCRBV各家族基因扫描和谱型分析.不同细胞亚群间数据比较采用配对t检验.结果 8例患者外周血淋巴细胞TCR BV家族CDR3谱型发生明显偏移,表现为单克隆性,寡克隆性及偏峰性克隆增生.8例患者CD8~+T淋巴细胞亚群发生TCR BV CDR3谱型偏移家族总个数高于去除CD8~+T淋巴细胞群的外周血单个核细胞[(10.6±4.7)个比(4.1±3.1)个,t=6.619,P<0.01)];比较单、寡克隆增生2种形式偏移的家族,CD8+T淋巴细胞亚群也高于代表CD4+T淋巴细胞的去除CD8~+T淋巴细胞群的外周血单个核细胞[(8.8±4.5)个比(3.9±2.8)个,t=5.706,P<0.01].对其中3例患者磁珠分选前后TCR BV谱型的比较发现,分选后CD8+T淋巴细胞亚群发生TCR BV谱型偏移家族数均高于分选前.结论 通过淋巴细胞亚群分选方式分析TCR BV家族CDR3谱型变化可以减少不同淋巴细胞亚群之间的峰型重叠干扰;应用这一方法分析外周血CD8~+T淋巴细胞亚群克隆增生程度有助于了解CHB患者炎症的发生机制.     

慢性乙型肝炎患者外周血T淋巴细胞受体谱系分析及互补决定区3序列测定

目的从新的角度探讨慢性乙型肝炎（CHB）炎症活动期外周血T淋巴细胞克隆性增生情况，了解T淋巴细胞免疫应答在CHB发病机制中的状态及作用。方法利用逆转录聚合酶链反应（RT-PCR）扩增T淋巴细胞受体（TCR）β链V区基因各家族（BV），并采用免疫指纹技术（immunoscope）对4名健康献血员及8例处于炎症活动期CHB患者外周血TCR BV家族基因的优势利用情况及克隆性增生T淋巴细胞β链互补决定区3（CDR3）序列进行分析。结果4名健康献血员外周血T淋巴细胞TCR BV家族CDR3谱型均呈正态分布，而8例CHB患者均出现一个或多个TCR BV家族单克隆或寡克隆性增生。对克隆性增生T淋巴细胞β链CDR3区序列测定证实存在不同的CDR3序列。结论CHB活动期外周血T淋巴细胞存在明显克隆性增生，进一步提示T淋巴细胞免疫应答可能参与CHB的发病过程，且可能有多个病毒抗原表位参与了细胞免疫应答。     

“荧光定量PCR溶解曲线法”监测HIV感染者TCR α链CDR3谱系漂移的研究

目的利用"荧光定量PCR溶解曲线分析技术"监测HIV感染者外周血T细胞TCRα链CDR3谱系漂移(单/寡/多克隆增生)。方法提取6例HIV感染者、4例健康者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的总RNA,逆转录成cDNA,设计人32个TRAV基因家族上、下游引物,荧光定量PCR(FQ-PCR)扩增32个TRAV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生。结果健康者外周血T细胞TCRα链32个家族CDR3表达频率不一致,各家族PCR产物的"溶解曲线谱型图"显示多克隆增生的高斯分布,呈现溶点不同的CDR3多态性,6例HIV感染者的外周血TCRα链CDR3谱系的32个家族CDR3表达频率不一致,患者各家族PCR产物的"溶解曲线谱型图"显示多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族。结论荧光定量PCR溶解曲线法监测人TCRα链CDR3谱系漂移的方法稳定、简便,可以用来监测健康者和HIV感染者外周血T细胞TCRα链CDR3谱系漂移。     

慢性乙型肝炎病毒感染者外周血T细胞受体β链V区谱型差异研究

目的 探讨外周血T细胞受体（TCR）β链V区（BV）CDR3区谱型差异在慢性HBV感染者炎症发生中的作用。方法研究对象为9例慢性HBV感染无症状携带（AsC）者（AsC组）和12例活动期慢性乙型病毒性肝炎（CHB）患者（CHB组）,分别采集其外周血,肝素钠抗凝,分离单个核细胞,提取RNA并应用RT-PCR扩增TCR BV的CDR3区基因24个家族,采用免疫指纹技术进行TCR BV各家族基因扫描和谱型分析。结果 两组外周血淋巴细胞TCR BV谱型均发生不同程度单克隆性、寡克隆性及偏峰性克隆增生,CHB组发生谱型偏移的TCR BV家族个数及CDR3谱型发生单克隆性和寡克隆性偏移家 族总数均显著多于AsC组（P〈0.05）。结论 CHB患者外周血T细胞克隆性增生程度高于AsC患者,此可能与慢性HBV感染者炎症的发生机制相关。     

类风湿关节炎患者T细胞抗原受体BV基因的限制性取用初步研究

目的 研究类风湿关节炎（rheumatoid arthritis，RA）患者病灶部位即关节滑膜组织浸润的受某种自身抗原驱动的T淋巴细胞抗原受体（T cell receptor，TCP）β链的取用格局。方法 采用实时定量聚合酶链反应（polymerase chain reaction，PCR）的方法分析RA患者病灶部位T淋巴细胞抗原受体β链的取用：采用PCR—SSP方法对所调查的RA患者的人类自细胞抗原（human leucocyte antigen，HLA）进行了分型。结果 研究发现中国人群RA患者病灶关节滑膜浸润的T淋巴细胞抗原受体主要取用BV14和BV16家族：中国人群RA患者HLA基因型主要以HLA DRBI＊0405为主要特征，与白种人RA患者不同。结论 研究提示中国人群RA患者病灶区自身反应性T细胞具有TCR BV14和BV16的限制性取用．其中TCR BV16的偏移为国内外首次报道。而HLADRBI＊0405类风湿关节炎患者表现出TCR BV16取用的趋势，提示TCR BV16取用受主要组织相容性复合体（MHC）的约束，这为进一步分析诱发RA的发生的免疫异常和自身反应性T细胞的生物学特陛提供了重要试验基础。     

合并肝细胞脂肪变性的慢性乙型肝炎患者调节性T淋巴细胞的变化及其意义

本研究旨在通过检测合并肝细胞脂肪变性的慢性乙型肝炎(CHB)患者外周血CD4+ CD25+T淋巴细胞水平与肝组织中Foxp3的表达,探讨肝细胞脂肪变性对CHB患者调节性T淋巴细胞(Tregs)的影响及其意义,现报道如下.     

染料法实时荧光聚合酶链反应检测人外周血αβT淋巴细胞克隆的条件优化及初步应用

目的 探讨实时荧光PCR检测人外周血αβT淋巴细胞克隆性扩增的反应条件及在监测慢性乙型肝炎(慢乙肝)患者外周血克隆特异性T淋巴细胞上的初步应用.方法 染料法(SYBR Green Ⅰ)实时荧光PCR对6例健康献血者外周血单个核细胞(PBMC)来源的T淋巴细胞受体β链可变区(TCRBV)基因进行扩增,分别就退火温度、引物浓度、循环数等进行比较研究.优化后PCR检测12例慢乙肝患者PBMC来源的TCRBV的24个基因家族,作PCR产物的熔解曲线峰形图,并分析T淋巴细胞克隆性扩增情况.结果 染料法实时荧光PCR检测 TCRBV基因家族的最佳退火温度为60.6℃,引物终浓度为0.5 μmol/L,循环数为40个,且熔解曲线分析起始温度80℃优于75℃.PCR产物熔解曲线峰形图上发现,慢乙肝患者某些TCRBV基因家族表现为单峰,测序结果表明其为单克隆PCR产物.结论 本研究优化了染料法实时荧光PCR检测TCRBV基因家族方法,熔解曲线峰形图可用于检测人外周血T淋巴细胞的克隆性扩增,并可能用于检测慢乙肝患者外周血克隆特异性T淋巴细胞.     

Analysis of the CDR3 length of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B.

To clarify the characteristics of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B (CHB), we investigated the patterns of Complementarity Determining Region3 (CDR3) length distribution for all 24 TCR BV gene families and 32 TCR AV gene family in the peripheral blood lymphocytes of two patients with CHB and two healthy controls by immunoscope spectratyping technique. We found that the profiles of CDR3 length distribution for all TCR AV and TCR BV family showed Gaussian distribution (the polyclonal TCR alphabeta T cells) in healthy controls, however, the restricted usage of TCR BV and TCR AV family (the oligoclonal TCR alphabeta T cells) has been monitored in two CHB patients, furthermore, the oligoclonal TCR alphabeta T cells showed the different CDR3 sequences and length, it might be correlated to the different epitope of hepatitis B virus (HBV) or the different HLA type of the patients. Detailed analysis of the CDR3 length of TCR alphabeta T cells might be interesting to light on the further study of the individualized immunotherapy of CHB and the further research of the new T lymphocyte epitope vaccine of HBV.     

Analysis of the CDR3 region of alpha/beta T‐cell receptors (TCRs) and TCR BD gene double‐stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia

Recently, numerous reports have highlighted the restriction of the CDR3 length of T‐cell receptor (TCR) beta chain in T‐cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo‐clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T‐lineage acute lymphoblastic leukemia (T‐ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T‐ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T‐ALL patients had the recombination signal sequence (RSS) 5′‐ and 3′‐breaks end in the TCR BD2 gene using a specialized ligation‐mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T‐ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T‐ALL vaccine, as well as in improving our understanding of healthy human T‐cell development.     

Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection,single-cell PCR,and CDR3-spectratype analysis

Clonal expansions of CD8+ T cells have been identified in muscle and blood of polymyositis patients by PCR techniques, including T cell receptor (TCR) complementarity-determining region (CDR)3 length analysis (spectratyping). To examine a possible pathogenic role of these clonally expanded T cells, we combined CDR3 spectratyping with laser microdissection and single-cell PCR of individual myocytotoxic T cells that contact, invade, and destroy a skeletal muscle fiber. First, we screened cDNA from muscle biopsy specimens by CDR3 spectratyping for expanded TCR beta chain variable region (BV) sequences. To pinpoint the corresponding T cells in tissue, we stained cryostat sections with appropriate anti-TCR BV mAbs, isolated single BV+ T cells that directly contacted or invaded a muscle fiber by laser-assisted microdissection, and amplified their TCR BV chain sequences from rearranged genomic DNA. In this way, we could relate the oligoclonal peaks identified by CDR3-spectratype screening to morphologically characterized microdissected T cells. In one patient, a large fraction of the microdissected T cells carried a common TCR-BV amino acid CDR3 motif and conservative nucleotide exchanges in the CDR3 region, suggesting an antigen-driven response. In several cases, we tracked these T cell clones for several years in CD8+ (but not CD4+) blood lymphocytes and in two patients also in consecutive muscle biopsy specimens. During immunosuppressive therapy, oligoclonal CDR3-spectratype patterns tended to revert to more polyclonal Gaussian distribution-like patterns. Our findings demonstrate that CDR3 spectratyping and single-cell analysis can be combined to identify and track autoaggressive T cell clones in blood and target tissue. This approach should be applicable to other inflammatory and autoimmune disorders.     

Analysis of the CDR3 region of alpha/beta T-cell receptors (TCRs) and TCR BD gene double-stranded recombination signal sequence breaks end in peripheral blood mononuclear cells of T-lineage acute lymphoblastic leukemia

Recently, numerous reports have highlighted the restriction of the CDR3 length of T-cell receptor (TCR) beta chain in T-cells infiltrating solid tumors and hematological malignancies. However, these studies ignored the restriction of CDR3 length of TCR alpha chain and few of them attempted to reveal the mechanisms of the oligo-clonal expansion of T cells in the tumors. The primary aims of this study were twofold to: (i) analyze the CDR3 length of TCR alpha and beta chain in peripheral blood mononuclear cells of T-lineage acute lymphoblastic leukemia (T-ALL); and (ii) discover the relationship between the clonality of T cells and the process of TCR rearrangement in peripheral T cells. To this end, we investigated the TCR BV and TCR AV family spectratypes of two T-ALL patients and healthy controls using the immunoscope spectratyping technique. We found that the spectratypes exhibited a Gaussian distribution in healthy controls. However, the TCR repertoires of the two patients were highly restricted in the number of different TCR BV and TCR AV family members present. Furthermore, we found that the peripheral blood mononuclear cells (PBMC) of two T-ALL patients had the recombination signal sequence (RSS) 5'- and 3'-breaks end in the TCR BD2 gene using a specialized ligation-mediated polymerase chain reaction, implying the ongoing recombination of the TCR beta gene. Analysis of the particular CDR3 length of TCR alpha/beta T cells might be helpful for further study of the individualized therapy of T-ALL. This information will also be helpful in exploring new immunological pathogenesis and facilitating the design of a T-ALL vaccine, as well as in improving our understanding of healthy human T-cell development.