基于TCR基因位点的FT-aCGH分析鉴定T-ALL中异常TCR Vβ-IgH重排

目的:鉴定T细胞-急性淋巴母细胞白血病(T-ALL)异常基因重排。方法:利用基于T细胞受体(TCR)基因位点的精细定位的寡核苷酸阵列比较基因组杂交(FT-aCGH)技术分析1例T-ALL样本与对照组基因组DNA的差异,了解7号和14号染色体TCRβ、TCRγ和TCRαδ位点上可能的断裂点位置,根据所提供的初步结果,根据断裂点涉及的基因序列设计特异引物,采用连接介导PCR(LM-PCR)和序列分析等方法分析与之发生重排的基因序列。结果:FT-aCGH结果显示所检测T-ALL样本中,各TCR基因位点都存在断裂点,经过LM-PCR和序列分析,以及利用PCR和特异引物检测基因组DNA结果确定了T-ALL克隆为Vα8-Jα22基因重排,并发现了7号染色体在TCRβ基因位点,Vβ20-1基因片段与14号染色体位于免疫球蛋白重链区基因位点的IgHVII-26-2基因片段发生异常重排,形成t(7;14)(q34;q32)染色体易位。结论:利用FT-aCGH和LM-PCR技术在1例T-ALL中发现了一种新的Vβ20-1-IgHVII-26-2异常重排,这种异常重排有可能可以作为该肿瘤克隆的生物标记物。     

T—ALL中两种新的TCRδ基因重排δRec151051—Jδ1和δRec151298—Jδ1

目的 分析T细胞急性淋巴细胞白血病（T-ALL）细胞中TCRδ基因新的重排形式及其在正常人外周血和胸腺细胞中的分布情况。方法 利用半巢式PCR扩增2例T-ALL、10例正常人外周血和7例正常人胸腺细胞DNA的TCRδ基因组片段,了解其重排情况,克隆性PCR产物进一步进行苷酸序列分析确定重排位置,并进一步经PT-PCR检测其重排基因的表达情况。结果 在2例T-ALL病人白血病细胞发现两种新的TCR δRec-Jδ1基因重排方式,称为δRec151051-Jδ1和δRec151298^-Jδ1,该新的TCR δRec基因重排同时见于大多数正常人外周血和胸腺。RT-PCR显示该两种TCR δRec-Jδ1基因重排的产物可见于2例T-ALL中,而正常人为阴性。结论 本研究发现两种新的TCR δRec151051-Jδ1和δRec151298-Jδ1 基因重排。该重排基因表达于T-ALL中,而正常人不表达该重排基因,提示该重排形式可能与T细胞白血病的形成有一定的关系。     

检测人TCR BD基因重排时RSS断裂末端的LM-PCR方法的建立

目的：建立检测人T细胞TCR BD基因（Diversity Gene）经历重排的连接介导PCR方法（Ligation．MediatedPCR，LM-PCR），为T细胞重排和疾病的关系研究提供基础。方法：在人TCRβ链BD1与BD25′端重组信号序列（RSS）前，BD1与BD23′端RSS后各设计两套用于巢式PCR引物；设计并合成和双链RSS平断裂末端相接的特殊接头（BW Linker），提取胸腺组织、正常人和急性T淋巴细胞白血病（T-ALL）外周血单个核细胞（PBMC）样本总DNA，和BW-linker连接，进行巢式PCR，PCR产物琼脂糖电泳分析，阳性产物做胶回收，并克隆测序鉴定。结果：在1例胸腺组织提取的总DNA中证实存在BD1和BD25′和3′的RSS断裂末端，在2例T-ALLs的PBMC中检测到BD25′和3′的RSS断裂末端，2例正常人PBMC中未能检测到RSS断裂末端，阳性的LM-PCR产物通过测序鉴定，和基因组中序列完全相符合。结论：1例胸腺组织和2例T-ALL的PBMC中检测到RSS断裂末端，提示TCRBD基因正经历重排，测序结果表明建立的监测TCR重排的LM-PCR方法可靠，可用于T细胞重排模型和相关疾病的机制研究。     

T-ALL中两种新的TCRδ基因重排δRec_(151051)-Jδ1和δRec_(151298)-Jδ1

目的分析 T细胞急性淋巴细胞白血病 ( T- AL L )细胞中 TCRδ基因新的重排形式及其在正常人外周血和胸腺细胞中的分布情况。方法利用半巢式 PCR扩增 2例 T- AL L、10例正常人外周血和 7例正常人胸腺细胞 DNA的 TCRδ基因组片段 ,了解其重排情况 ,克隆性 PCR产物进一步进行核苷酸序列分析确定重排位置 ,并进一步经 RT- PCR检测其重排基因的表达情况。结果在 2例 T- AL L 病人白血病细胞发现两种新的 TCRδRec- Jδ1基因重排方式 ,称为 δRec1 51 0 51 - Jδ1和δRec1 51 2 98- Jδ1,该新的 TCRδRec基因重排同时见于大多数正常人外周血和胸腺。 RT- PCR显示该两种 TCRδRec- Jδ1基因重排的产物可见于 2例 T- AL L中 ,而正常人为阴性。结论本研究发现两种新的 TCRδRec1 51 0 51 - Jδ1和δRec1 51 2 98- Jδ1基因重排。该重排基因表达于 T- AL L中 ,而正常人不表达该重排基因 ,提示该重排形式可能与 T细胞白血病的形成有一定的关系     

正常人T细胞和胸腺细胞中多种新的TCRδRec基因重排

利用巢式或半巢式PCR扩增10例正常人外周血单个核细胞(PBMC)、4例分选CD3~+细胞和7例正常胸腺细胞DNA中TCR δRec区与Jδ1、Dδ3和Ja重排的基因片段,分析正常人外周血T细胞和胸腺细胞中TCR δRec基因重排情况。克隆性PCR产物进一步进行核苷酸序列分析确定其重排位置。结果发现了4种新的TCR δRec重排,包括TCR δRec_(149321)-Jδ1、TCRδRec_(149820)-Jδ1、TCR δRec_(151657)(Nx)-Ja和TCR δRec_(153199)-Jδ1等,其中以TCR δNx的重排最多见,通过利用不同模板DNA的PCR分析发现δRec重排在外周血和胸腺细胞中有所不同。结果显示TCR δNx-Jδ1重排在成熟和不成熟T细胞发生频率均较高,而TCR δNx-Dδ3重排在不成熟T细胞中发生率较高。但所有重排均不表达于mRNA中。本研究结果为TCR δ基因重排的研究补充了一些新的数据。     

正常人外周血和胸腺T细胞中TCR δRec151051-Jδ1和δ Rec151298-J δ 1基因重排的分布特点

目的分析正常人外周血和胸腺细胞中TCRδRec151051-Jδ1和δRec151298-Jδ1基因重排分布特点.方法利用半巢式和巢式PCR扩增10例正常人外周血单个核细胞(PBMCs)、4例分选的外周血CD3+T细胞和7例正常人胸腺细胞DNA的TCRδRec基因与Jδ1、Dδ3和ψJα重排的基因片段,从不同含量DNA的PCR分析,了解其重排分布频率.结果TCRδRec151051分别与Jδ1、Dδ3和ψJα的重排和TCRδRec151298-Dδ3重排见于大多数外周血T细胞和胸腺细胞中,而TCRδRec151298与Jδ1和ψJα的重排则多见于胸腺细胞中.对不同含量的DNA的PCR分析显示δRec重排的分布频率在外周血和胸腺细胞有所不同.结论TCRδRec151051、TCRδRec151298与Jδ1、Dδ3和ψJα的重排均存在于多数正常人T细胞中,TCRδRec151051-Dδ3最常见于成熟和未成熟T细胞中,而TCRδRec151298-Jδ1和TCRδRec151298-ψJα则主要见于未成熟T细胞中.     

正常人外周血和胸腺T细胞中TCR δRec151051—Jδ1和δRec151298—Jδ1基因重排的分布特点

目的 分析正常人外周血和胸腺细胞中TCR δRec151051-Jδ1和δRec151298-Jδ1基因重排分布特点。方法 利用半巢式和巢式PCR扩增10例正常人外周血单个核细胞（PBMCs）、4例分选的外周血CD3^ T细胞和7例正常人胸腺细胞DNA的TCR δRec基因与Jδ1、Dδ3和ψJa重排的基因片段,从不同含量DNA的PCR分析,了解其重排分布频率。结果 TCRδRec151051分别与Jδ1、Dδ3和ψJa的重排和TCR δRec151298-Dδ3重排见于大多数外周血T细胞和胸腺细胞中,而TCR δRec151298与Jδ1和ψJa的重排则多见于胸腺细胞中,对不同含量的DNA的PCR分析显示δRec重排的分布频率在外周血和胸腺细胞有所不同。结论 TCR δRec151051、TCR δRec151298与Jδ1、Dδ3和ψJa的重排均存在于多数正常人T细胞中,TCR δRec151051-Dδ3最常见于成熟和未成熟T细胞中,而TCR δRec151298-Jδ1和TCR δRec151298-ψJa则主要见于未成熟T细胞中。     

T-ALL患者外周血TCRγ/δ T细胞的分布和增殖特点

目的:了解T细胞急性淋巴白血病(T-cell acute lymphocytic leukemia,T-ALL)患者外周血TCR Vγ和Vδ亚家族T细胞的分布和克隆情况。方法:利用RT-PCR扩增9例T-ALL患者和10例正常人外周血单个核细胞中3个TCR Vγ和8个Vδ亚家族基因的互补决定区3(CDR3),了解TCR亚家族Vγ和Vδ亚家族细胞的分布;阳性产物进一步经基因扫描分析CDR3长度,从而了解其克隆性。结果:9例T-ALL患者3个Vγ亚家族的表达率都明显低于正常人Vγ亚家族的表达率(P<0.05);Vδ亚家族中只有Vδ1和Vδ3的表达率低于正常人(P<0.05)。3例T-ALL患者出现克隆性增殖的γ/δT淋巴细胞。结论:T-ALL患者外周血TCR Vγ和部分Vδ亚家族谱系出现限制性表达和克隆性增殖。同时存在克隆性增殖γδ T细胞提示可能为γδ 白血病性T细胞克隆。     

监测TCR CDR3漂移的免疫扫描谱型分析技术的建立与鉴定

外周血95％ T淋巴细胞的TCR由α（胚系基因中AV、AJ、AC重排）、β（胚系基因中的BV、BD、BJ、BC重排）两条多肽链组成，不同V（D）J重排后，由V基因末端、J基因前端和V-J（或V—D和D—J）连接时中间插入的核苷酸序列，分别组成了特异的TCRα和8的CDR3基因谱型的多样性。一种CDR3序列代表一个T细胞克隆，测定特定CDR3序列出现的频率可以反映特定T细胞克隆扩增的程度和功能状态。     

利用T细胞受体变异β基因谱系分析T细胞急性淋巴细胞白血病病人T细胞克隆性

目的:分析T细胞-急性淋巴细胞白血病(T-ALL)病人的T细胞克隆性.方法:利用RT-PCR方法分析6例T-ALL和10例正常人外周血单个核细胞中24个T细胞受体变异β(TCR Vβ)基因的CDR3长度,PCR产物再进一步进行基因扫描和序列分析.结果:3例病人的某些TCR Vβ亚家族T细胞呈单克隆或寡克隆性增殖,主要为Vβ2、3、6、9、21和24.其它3例及正常人均表现为多克隆性增殖T细胞.结论:部分T-ALL来自于TCR Vβ亚家族克隆性增殖T细胞.该方法有助于临床上检测微小残留病变.     

利用T细胞受体变异β基因谱系分析T细胞急性淋巴细胞白血病病人T细胞克隆性

目的:分析T细胞-急性淋巴细胞白血病(T-ALL)病人的T细胞克隆性。方法:利用RT-PCR方法分析6例T-ALL和10例正常人外周血单个核细胞中24个T细胞受体变异β(TCR Vβ)基因的CDR3长度,PCR产物再进一步进行基因扫描和序列分析。结果:3例病人的某些TCR Vβ亚家族T细胞呈单克隆或寡克隆性增殖,主要为Vβ2、3、6、9、21和24。其它3例及正常人均表现为多克隆性增殖T细胞。结论:部分T-ALL来自于TCR Vβ亚家族克隆性增殖T细胞。该方法有助于临床上检测微小残留病变。     

A case of CD3+, CD4-, CD8-, WT31- acute T-cell leukemia

The article reports a case of acute T-cell leukemia (T-ALL) with the unusual CD3+, WT31 phenotype. On surface marker analysis, the blast cells were found to be CD7+, CD2-, CD5-, CD1-, CD4-, CD8-, CD3+ and negative for B-lymphoid and myeloid lineage. The cells had both TCR beta and TCR gamma gene rearrangement but had negative results with the monoclonal antibody WT31, which reacts with a framework epitope on the T alpha/beta heterodimer (Ti) of the conventional T-cell receptor (TCR). This suggests an association of the CD3 molecular complex with a different polypeptide (the alternate TCR gamma). In two similar reported cases of T-ALL (WT31-, CD3+, CD4-, CD8-), the CD3 molecular complex was found to be associated with a product of the T gamma gene.     

Rearrangements of t-cell receptor β-chain genes in human leukaemias

Rearrangements of the T-cell receptor (TCR), β-chain genes and immunoglobulin (Ig) heavy chain genes in several T-cell leukaemias (T-ALL and ATL), and some B-cell and myelogenous leukaemias were investigated. Two out of 15 cases of T-cell leukaemia tested failed to show a rearrangement pattern of TCR β genes although both expressed mRNA for this gene. The remaining 13 cases showed diverse patterns of rearrangements involving either Cβ₁, Cβ₂ or both. Cβ₁, but not Cβ₂ was deleted in s the T-cell leukaemias. Polyclonal T cells from four normal individuals showed the germ line pattern and an additional two bands in Hind III digested DNA. Except for one, all cases of C-ALL (B-cell leukaemia) showed a rearranged J_H locus which was not evident in any of T-cell leukaemias studied. One case of B-cell leukaemia showed a rearrangement of both TCR β genes and J_H genes. The results of these studies suggest that rearrangement of TCR and Ig genes occurs at a very early stage of differentiation of stem cells and does not appear to play a direct role in leukaemogenesis per se.     

Multipoint interphase FISH in childhood T-acute lymphoblastic leukemia detects subpopulations that carry different chromosome 3 aberrations

We examined chromosome 3 in 32 childhood acute lymphoblastic leukemia (ALL) bone marrow samples. Using interphase multipoint FISH (mp-FISH), which was developed by our group, with 42 chromosome 3-specific probes, we detected clonal chromosome 3 aberrations in 4 T-cell ALL (T-ALL) cases. Four out of seven T-ALL cases carried 3q trisomies. One T-ALL case carried either trisomy 3 (in 15% of the cells) or a 23-megabase (Mb) 3p13 approximately p12 deletion in a different subpopulation of cells of 32%. Another T-ALL case had either 3q trisomy in 11% or a 12-Mb 3p12 approximately p13 deletion in 19% of the cells. The deletions were overlapping. In both cases, the majority of the bone marrow cells (47 and 70%, respectively) were normal chromosome 3 disomics. The interstitial deletions detected harbor a known homozygous deletion region between 72.6 and 78.8 Mb, which has been described in lung and breast tumors and contains the DUTT1/ROBO1 tumor suppressor gene. These deletions detected by mp-FISH would have remained unnoticed by conventional cytogenetics and multiplex FISH, as well as by current methods based on total tumor DNA analysis such as comparative genomic hybridization (CGH), array CGH, and loss of heterozygosity (LOH).     

Exogenous Thy-1.1 expression as a marker for thymocyte maturation in transgenic mice.

We established a line of transgenic mice carrying the exogenous mouse Thy-1.1 gene (8.2 Kb Eco RI genomic DNA fragment). In these mice, Thy-1.1 was expressed on thymocytes but not on peripheral T cells, presumably due to the lack of cis-acting element(s) on the microinjected genomic DNA. Even in the thymus, however, while most of the CD3/TCR- thymocytes were positive for the transgenic Thy-1.1 gene expression, the CD4+ or CD8+ single positive (SP) thymocytes were composed of two groups, one Thy-1.1+ and one Thy-1.1-, suggesting that the former belongs to cells at premature stages of terminal T cell differentiation. There was no difference in the amount of CD3/TCR complexes expressed on two such SP thymocyte subsets. In the double negative (DN) thymocytes, all the CD3/TCR+ cells were Thy-1.1-. These results suggest that the maturational process of CD3+ DN thymocytes differs from that of SP thymocytes. The unique distribution of Thy-1.1+ population among CD3+ thymocytes suggests that the transgenic Thy-1.1 gene expression can serve as a useful marker to examine terminal maturation processes of CD3+ thymocytes.     

多发性骨髓瘤患者外周血中TCR Vβ亚家族nave T细胞水平变化

目的： 检测多发性骨髓瘤（MM）患者外周血中23个TCR Vβ亚家族的T细胞受体删除DNA环（sjTRECs）的存在特点，从而了解MM患者相应Vβ亚家族nave T细胞的近期胸腺输出情况。方法: 利用半巢式PCR分别扩增12例MM患者每5×104个外周血单个核细胞（PBMCs）中的23个Vβ亚家族sjTRECs，10例正常人外周血作为对照。结果: 在5×104个PBMCs中，检测到MM患者sjTRECs的Vβ亚家族数量约为（5.00±2.45）个，与正常人的（9.60±5.48）个相比，检出的亚家族数量明显减少（P<0.05）；23个Vβ亚家族sjTRECs在正常人中均可以检测到，而在MM患者仅检测到部分；并且Vβ2、Vβ10、Vβ16、Vβ17和Vβ21等5个亚家族sjTRECs的检出率明显低于正常水平。12例MM患者检出的亚家族数量（2-9个）不等，患者的年龄与检出的亚家族数量存在负相关（r=-0.892；P<0.01）。结论: MM患者胸腺近期输出的23个Vβ亚家族nave T细胞存在不同程度的缺失或水平降低，表明患者存在不同程度的细胞免疫功能缺陷以及T细胞谱系重建的能力和潜能受损。     

Expression of the alpha/beta and gamma/delta T-cell receptors in 57 cases of peripheral T-cell lymphomas. Identification of a subset of gamma/delta T-cell lymphomas.

Fifty-seven cases of peripheral T-cell lymphoma were studied for cell expression of the T-cell receptor (TCR) chains, using monoclonal antibodies specific for the beta chain (beta F1) of the alpha/beta TCR, and for the delta chain (anti-TCR delta-1) of the gamma/delta TCR. Three different patterns were demonstrated: in 39 cases (69%), the phenotype (CD3+beta F1+TCR delta-1-) was that of most normal T cells. A second pattern was found on six cases (10%), which were of CD3+beta F1-TCR delta-1+ phenotype, and in which DNA analysis showed a clonal rearrangement of the delta locus in the five cases studied. It is suggested that these cases are the neoplastic counterpart of the small subpopulation of normal T cells that express gamma delta receptor. It is of considerable interest that these gamma delta lymphomas had unusual clinicopathologic presentations, as one case corresponded to a lethal midline granuloma and the five others to hepatosplenic lymphomas with a sinusal/sinusoidal infiltration in spleen, marrow, and liver. The fact that the distribution of the neoplastic gamma delta cells in the splenic red pulp resembles that of normal gamma delta cells reinforces the concept of a preferential homing of gamma delta T cells to this tissue. A third pattern (CD3 +/- beta F1-TCR delta-1-) was seen in 12 cases (21%), in which, by contrast to normal post-thymic T cells, no evidence of either alpha beta or gamma delta T cell receptor was found.