TCR Vβ7基因转染PBLs的表达及抗肝癌的作用

目的 :将TCRVβ7基因转化到淋巴细胞后研究肝癌细胞的生物学作用。 方法 :用RT PCR的方法扩增出TCRVβ7基因 ,克隆到表达载体pLXSN上 ,用脂质体转染的方法 ,将重组载体导入淋巴细胞 ,然后将淋巴细胞作用于肝癌细胞 ,淋巴细胞用流式细胞仪测表型和用激光共聚焦检测TCRVβ7 1基因的表达 ,肝癌细胞作超微结构分析。 结果 :TCRVβ7基因表达的跨膜蛋白显著增多 ,而且淋巴细胞增多 (P <0 0 5 ) ,肝癌细胞出现凋亡。结论 :TCRVβ7亚家族具有识别肝癌抗原的作用并能激活T细胞的杀伤肝癌细胞的功能。

苯接触工人外周血T细胞受体TCRV_β亚家族基因多态性分析

目的 了解苯接触工人外周血TCRVβ 亚家族T细胞的分布及其克隆性 ,寻找防治苯中毒的免疫指标。方法 采用RT PCR扩增 6例不同工龄低浓度苯接触工人外周血单核细胞的TCRVβ2 4个亚家族的互补决定区 3(CDR3) ,产物进一步经基因扫描分析确定T细胞的克隆性 ,并与 8名健康人作对照。结果 8名健康者表达全部Vβ 亚家族 ,PCR产物呈多峰图像 (多克隆 )。苯接触工人随工龄不同 ,外周血损伤的情况不同而出现Vβ 表达减少 ,PCR产物出现寡克隆或双克隆改变。结论 低浓度苯接触工人的外周血T细胞的TCRVβ 亚家族表达出现限制性 ,部分Vβ 亚家族呈克隆性增殖 ,可能由于苯损害T细胞而造成机体免疫的不同改变。

Stable clonal expansion of the T-cell receptor Vβ6, Vβ17 and Vβ19 T cells in a cGVHD case using genescan analysis

Objective To investigate the distribution and clonality of the T-cell receptor (TCR) Vβ repertoire in chronic graft versus host disease (cGVHD).Methods The complementarity determining region 3 (CDR3) of the TCRβ gene with 24 variable regions was amplified in peripheral blood mononuclear cells drawn from one cGVHD patient after allogenic bone marrow transplantation (allo-BMT) 35, 39, 43 or 45 months respectively, using RT-PCR, to observe the expression of TCR Vβ repertoire T cells. The PCR products were further analyzed by genescan to evaluate clonality of T cells. Ressults Fourteen or 16 TCR Vβ subfamily T ceils were detected in each sample of cGVHD case. Oligoclonal T cells were identified in TCR Vβ 6, 16, 17, 19 and 21 subfamilies. The stable clonal T cells in all samples were identified in Vβ6, Vβ17 and Vβ21 subfamilies.Conclusion Skewing distribution and stable clonal expansion of T cells can be found in cGVHD cases and it may be related to the initiation of cGVHD.

CⅡ诱导RA患者关节滑液中自身反应性T细胞的免疫学特性分析

目的 :探讨CⅡ诱导的RA患者关节滑液中自身反应性T细胞免疫学特性。方法 :分离RA患者关节滑液中SFMC ,用CⅡ刺激后分析反应性的T细胞频率 ;用ELISA方法检测CⅡ刺激SFMC后上清中的IFN γ和IL 4的含量及通过流式细胞检测SFMC表面标志的表达 ;应用半定量PCR分析外周血及滑膜液中TCRVβ基因表达格局。 结果 :SFMC中CⅡ反应性的T细胞频率高于PBL ;IFN γ的含量在CⅡ刺激SFMC后上清中较高 ,而在未刺激中较低 (P <0 0 1) ,在PBL中更低 (P <0 0 5 ) ;SFMC上CD8及CXCR3表达多于PBMC(P <0 0 5 ) ;外周血T细胞表达绝大多数Vβ基因 ,而滑膜液中TCRVβ基因取用呈现明显的不均衡性。结论 :CⅡ反应性T细胞在SFMC中占优势 。

Changes in the T-cell receptor Vβ gene repertoire after allogeneic hematopoietic stem cell transplantation

Background We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) Vβ gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR Vβ genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.Results The 24 TCR Vβ gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Anoth er 4 donors expressed part of the 24 TCR Vβ subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR Vβ subfamilies were skewed and restric ted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR Vβ subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6-30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in Vβ 2-3, 16-17, 18-19, 21 and Vβ 23 in patients with GVHD and in Vβ 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had Vβ 15 and 16 T cell expansion after transplantation. One patient displayed Vβ 18 T cell expansion after donor lymphocyte infusion (DLI).Conclusions Normal individuals express the entire 24 TCR Vβ ge ne repertoire and have polyclonal distribution. However, the TCR Vβ gene repertoire is only partially expressed in some donors. The TCR Vβ gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR Vβ gene subfamilies increases over time post- transplantation. GVHD and GVL effects may induce the proliferation of T cell clones.Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.

T cell receptor Vβ repertoire usage and clonal expansion of T cells in chronic myelogenous leukemia

Background In general,it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know,few studies have dealt with the distribution of oligoclonal T cells in leukemia,so we investigated the distribution and clonality of TCR Vβ repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase. Methods The complementarity determining region 3 (CDR3) of TCR Vβ24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vβ repertoire,the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vβ T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.Results The expression pattern of TCR Vβ repertoire in different individuals are different. Vβ2-21 subfamilies could be detected in CML cases. The frequent usage Vβ repertoire in CML was Vβ1,Vβ2 orVβ13. Most of the PCR products from 27 patients displayed polyclonality,while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vβ16 subfamilies. The frequent usage of Vβ genes in clonal expansion was Vβ3,Vβ13 or Vβ21. Multiple Vβ clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vβ21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.Conclusions These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vβ3,Vβ13 and Vβ21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.