实时定量PCR检测正常人外周血T细胞和胸腺细胞中sjTRECs水平

目的了解正常人外周血T细胞和胸腺细胞中信号结合T细胞受体删除 DNA环(sjTRECs)的含量,从而推测正常人中幼稚T细胞的含量和胸腺的输出功能. 方法利用实时定量PCR和TaqMan方法检测11例正常人外周血单个核细胞、7例儿童和3例成人胸腺细胞DNA中sjTRECs的水平. 结果正常人中sjTRECs含量分别为:8.83±4.81/1000个外周血单个核细胞;27.31±3.23/1000个成人胸腺细胞和170 .29 ±59.52/1000个儿童胸腺细胞. 结论 sjTRECs水平与年龄有关,正常人外周血中每1000个单个核细胞中约含4.5个幼稚T细胞.     

实时荧光定量PCR分析脐血T细胞亚群中TRECs水平

近期研究发现成人胸腺仍存在活化T细胞合成的功能，胸腺近期功能需要通过测定胸腺近期输出的幼稚(Naive)T细胞的数量来衡量。而能够作为Naive T细胞的标志的是T细胞受体删除DNA环(T-cell receptor excision DNA circles，TRECs)。我们的前期研究也提供了正常人外周血T细胞和胸腺细胞中TRECs含量的情况，本研究进一步分析脐血中不同组分T细胞的TRECs含量特点。     

TCR Dβ-Jβ sjTRECs检测方法的建立和应用

目的：建立检测TCR Dβ-Jβ sjTRECs的方法，并了解不同T细胞受体Dβ-Jβ之间T细胞受体删除DNA环(sjTRECs)在胸腺细胞和外周血T细胞中的形成情况。方法：利用巢式PCR分别扩增10例正常人外周血单个核细胞和3例正常胸腺细胞DNA中不同的Dβ片段和Jβ重排时形成的sjTRECs的情况，PCR产物进一步进行克隆和序列分析以确定结合区的位置。结果：可检测到Dβ1与5个Jβ1基因片段、Dβ2与4个Jβ2基因片段分别形成的sjTRECs，其中以Dβ1-Jβ1S1、Dβ1-Jβ1S2和Dβ2-Jβ2S2sjTRECs最常见，PCR产物经克隆和序列分析证实其形成Dβ-jβsjTRECs的情况。结论：成功地建立了检测Dβ-Jβ-sjTRECs的方法，为分析各TCRβ亚家族的sjTRECs含量和确定TCRβ naive细胞提供了新方法。     

CD4T细胞缺乏对胸腺细胞凋亡影响的研究

通过对ＬＥＣ大鼠和正常对照鼠胸遥细胞的凋亡的研究，探讨了ＣＤ４Ｔ细胞缺乏对胸腺细胞凋亡的影响。电子显微镜，抗ｓｓ－ＤＮＡ抗体免疫细胞化学染色及ＤＮＡ片段梯形电泳带结果提示：ＣＤ４Ｔ细胞缺乏可诱导不成熟Ｔ细胞凋亡，对成熟Ｔ细胞无明显影响。     

实时定量PCR检测白血病中BCL11B基因的表达水平

目的建立BCL11B基因的定量检测方法,并分析其在白血病中的表达水平。方法利用实时定量RTPCR分析TALL(12例)、BALL(8例)、BCLL(6例)、AML(7例)和正常对照(10例)外周血单个核细胞(PBMC)中BCL11B基因的表达水平,以β2微球蛋白(β2M)基因表达水平作为内对照。结果TALL患者PBMC中BCL11B表达水平(553.84±564.01拷贝105β2M拷贝)明显高于正常对照和其他白血病患者(P=0.006,P=0.013,P=0.031,P=0.020);而AML组BCL11B表达水平(0.02±0.04拷贝105β2M拷贝)则明显低于正常对照组(P=0.000)、BALL组(P=0.006)和TALL组(P=0.020);BALL(1.99±1.59拷贝105β2M拷贝)和BCLL(2.26±3.57拷贝105β2M拷贝)中BCL11B表达水平与正常对照(2.20±1.01拷贝105β2M拷贝)无显著差别。结论建立了实时定量RTPCR检测BCL11B方法,TALL中BCL11B高表达可能与其发病有一定关系。     

定量检测T细胞受体重排删除DNA环含量评价急性非淋巴细胞白血病患者胸腺近期输出功能

目的检测急性非淋巴细胞白血病(ANLL)患者T细胞受体重排删除DNA环(signaljoint T-cell receptor exc ision DNA c irc les sjTRECs,TRECs)的含量,从而探讨患者的初始型naive T细胞水平和胸腺近期输出功能特点。方法利用实时定量PCR(TaqM an)方法,检测77例ANLL患者外周血单个核细胞(PBMC)TRECs的水平,并根据外周血中CD3阳性率计算CD3细胞中TRECs水平。14例ANLL缓解期患者和14例正常人外周血作为对照。结果ANLL患者外周血中TRECs含量为(0.43±0.92)拷贝/1 000 PBMCs,(2.02±3.25)拷贝/1 000 CD3+细胞,明显低于正常人TRECs水平[(4.10±3.65)拷贝/1 000 PBMCs和(6.84±4.71)拷贝/1 000 CD3+细胞,P=0.0000和P=0.0001]和缓解期患者[(2.19±2.49)拷贝/1 000 PBMCs和(6.30±7.13)拷贝/1 000 CD3+细胞,P=0.0000和P=0.0005]。各亚型ANLL患者的TREC水平差异无统计学意义(P>0.05)。结论绝大多数ANLL型患者胸腺近期输出naive T细胞功能明显降低,缓解期患者的胸腺近期输出功能有一定程度恢复。     

小鼠胸腺树突状细胞系MTSC4诱导早期T细胞株C320…

通过流式细胞仪分析了我室自建的小鼠胸腺树突状细胞系ＭＴＳＣ４与小鼠肿瘤性早期Ｔ细胞株Ｃ３２０细胞共育后，Ｃ３２０细胞表达ＩＬ－２Ｒ的状态，分析了ＣｏｎＡ和ＰＨＡ对Ｃ３２０及与ＭＴＳＣ４共育后的Ｃ３２０表达ＩＬ－２Ｒ的诱导促进作用以及ＩＬ－２Ｒ＾＋Ｃ３２０细胞在脱离外源性刺激后其比例的变化，ＭＴＳＣ４及ＣｏｎＡ和ＰＨＡ在不同程度上抑制Ｃ３２０细胞的无限制增殖，ＣｏｎＡ和ＰＨＡ对与ＭＴＳＣ４共育后的Ｃ     

实时荧光定量PCR检测幼儿腹泻腺病毒

目的:建立实时荧光定量PCR方法,并检测腺病毒Ad40或Ad41感染的粪便标本。方法:用T-A克隆技术构建含腺病毒基因的载体作为标准模板,采用Taqman探针标记技术,建立实时荧光定量PCR方法。采集幼儿腹泻标本248例,分别用直接免疫荧光法和实时荧光定量PCR检测腺病毒Ad40和Ad41,比较两种方法的阳性检出率。结果:实时荧光定量PCR灵敏度和准确度(95.60%和96.80%)均高于直接免疫荧光法(78.5%和82.40%)(P0.05),而两者特异度差异无统计学意义(97.30%vs 96.70%,P0.05)。248例待检样本,直接免疫荧光法检出率为2.42%(6/248),实时荧光定量PCR检出率为7.66%(19/248),明显高于直接免疫荧光法(χ2=3.675,P0.05)。结论:成功建立实时荧光定量PCR检测腺病毒Ad40或Ad41方法。其灵敏度、准确度和阳性检出率经均优于直接免疫荧光法,且简便快速。     

地塞米松及CD4T细胞缺乏对胸腺细胞Apoptosis影响的再观察

地塞米松及ＣＤ４Ｔ细胞缺乏对胸腺细胞Ａｐｏｐｔｏｓｉｓ影响的再观察姜玉珍张凤春林玉梅张秀梅（白求恩医科大学第三临床学院，长春１３００３１）王志申（吉林省人民医院，长春１３００２１）中国图书分类号Ｒ９７７．１１Ｒ９６７细胞凋亡（Ａｐｏｐｔｏｓｉｓ）在胸...     

人外周血sjTREC水平与年龄的相关性调查

目的探讨人外周血中sjTREC水平及其与年龄的相关性。方法收集225例不同年龄健康人外周血样品,抽提基因组DNA,利用实时定量PCR技术,定量测定不同年龄组外周血中sjTREC含量的分布情况。sjTREC含量与年龄的关系作相关分析;各年龄组性别差异作t检验。结果 225例外周血sjTREC含量随年龄增长逐渐减少;对0～14岁、15～54岁及55岁以上3组进行方差分析(LSD检验),差异显示有统计学意义(P0.001);性别差异不显著;供体年龄与外周血sjTREC水平呈显著负相关,r=-0.823(P0.001)。结论人外周血中sjTREC含量与年龄存在明显相关性,对于法医物证实践中血痕的年龄推测具有一定指导意义。     

Altered naive CD4 and CD8 T cell homeostasis in patients with relapsing-remitting multiple sclerosis: thymic versus peripheral (non-thymic) mechanisms

We have reported previously that naive T cells from relapsing-remitting multiple sclerosis (RRMS) patients have T cell receptor (TCR) repertoire shifts, but the basis of these TCR repertoire shifts was uncertain. Here, we questioned whether RRMS patients have altered naive CD4 and CD8 T cell homeostasis by studying homeostatic proliferation and thymic production in RRMS patients and healthy controls. We measured thymic production by quantifying signal joint T cell receptor excision circles (sjTRECs). Both naive T subsets from controls showed an age-associated decrease in sjTRECs, i.e. evidence of progressive thymic involution, but we detected no age-associated decrease in sjTRECs in RRMS patients. Instead, naive CD8 T cells from patients had lower sjTRECs (P = 0.012) and higher Ki-67 proliferation levels (P = 0.04) than controls. Naive CD4 T cell sjTRECs did not differ between patients and controls. However, in RRMS these sjTRECs correlated strongly with CD31, a marker expressed by newly generated CD4 T cells but not by naive CD4 T cells that have undergone homeostatic proliferation. HLA-DR2 positivity correlated negatively with naive CD4 T cell CD31 expression in RRMS (P = 0.002). We conclude in RRMS that naive T subsets have homeostatic abnormalities due probably to peripheral (non-thymic) mechanisms. These abnormalities could have relevance for MS pathogenesis, as naive T cell changes may precede MS onset.     

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。     

Real-time PCR分型法检测苏南地区汉族人群KIR基因多态性

目的利用SYBR Green I Real-time PCR分型方法检测杀伤细胞免疫球蛋白样受体(killer cell immunoglobulin-likereceptor,KIR)基因,探讨苏南地区汉族人群KIR基因的分布特点。方法应用SYBR Green I Real-time PCR法对191名苏南地区汉族非亲缘健康人群进行KIR基因分型。结果 SYBR Green I Real-time PCR法有效地进行了KIR基因分型。已知的16种KIR基因在苏南地区汉族人群均被检出。框架基因2DL4、3DL2、3DL3和假基因3DP1存在于所有受检个体中。最常见的非框架基因为2DL1、2DL3、3DL1、2DS4以及假基因2DP1。共检出33种KIR基因型,最常见的为AA1(39.27%),其次为BX2、BX4和BX8。发现仅在新加坡华人报道的罕见基因型BX331和BX337,及仅在墨西哥人群罕见的基因型BX427。结论苏南汉族人群中检测出已知的16种KIR基因,共发现33种基因型,最常见的为AA1,并见到3个罕见基因型BX331、BX337和BX427。     

SEB活化的人外周血T细胞CD25,CD69的表达

本文用超抗原葡萄球菌肠毒素B(SEB)诱导外周血淋巴细胞增殖。结果显示出微量超抗原能诱导外周血淋巴细胞的增殖,大量的增殖反应发生在SEB刺激后的第5天,增殖的细胞是CD4~+T细胞,它们由刺激前的27％增加到42％。外周血活化的T细胞不依赖外源性IL-2:大量内源性IL-2的存在抑制T细胞的增殖反应。伴随CD4~+T细胞的增殖,CD25和CD69分子表达明显增加。提示SEB能改变外周血T细胞表面的分子表达。     

Real-time PCR method for the quantitative analysis of human T-cell receptor gamma and beta gene rearrangements

Analyzing the status of T-cell receptor (TCR) gene rearrangements has been an essential part of deciphering the stages of thymocyte development, understanding the β vs. γδ lineage decision, and characterizing T-cell leukemias. Methods such as PCR and quantitative Southern blotting provide useful information, but also have significant shortcomings such as lack of quantitation in the case of PCR and technical challenges in the case of Southern blotting. Here we describe a real-time PCR method that overcomes many of these shortcomings. This new method shows comparable results for the fraction of unrearranged TCRγ and TCRβ genes in human thymocytes and peripheral blood T cells as Southern blotting, and has the advantages of being simple to perform, highly quantitative, and requiring nanogram quantities of DNA. We also describe a real-time PCR method to quantitate T-cell receptor excision circles formed during TCRβ rearrangements.     

T Cell Receptor Excision Circles (TRECs) in Relation to Acute Cardiac Allograft Rejection

The purpose of this study was to quantify T cell receptor excision circles (TRECs) in blood mononuclear cells of cardiac transplant recipients and to investigate a possible relationship between TREC levels and rejection episodes. In addition, we investigated the correlation of TREC levels with age and also compared the levels between transplant recipients and healthy individuals. TREC levels were assessed by quantitative competitive PCR in 70 blood samples from 27 graft recipients and in 66 blood samples from 66 healthy individuals. The results showed: (1) higher TREC levels during rejection than during rejection-free periods (medians 9.0 vs. 0.3; p<0.001); (2) no suggestion of correlation with doses of prednisone or time after transplantation; (3) a negative correlation between TREC levels and age; and (4) lower TREC levels in cardiac recipients than in age-matched healthy controls. The value of blood TREC level measurements as an approach to rejection monitoring warrants future investigation.     

桥本甲状腺炎患者外周血滤泡辅助T细胞水平及临床意义

目的 检测桥本甲状腺炎(HT)患者外周血滤泡辅助性T细胞(follicular helper T cells,Tfh)水平,探讨Tfh与HT发病的关系.方法 采用电化学发光法检测HT患者及对照组外周血FT3、FT4、TSH、甲状腺自身抗体水平,采用流式细胞术分析各组试验对象的外周血Tfh的水平,同时进行相关性分析.结果 与对照组相比,桥本甲状腺炎患者外周血Tfh比例显著升高(P＜0.05);桥本甲状腺炎患者外周血Tfh与甲状腺自身抗体TPO-Ab呈正相关(P＜0.05),但与Tg-Ab无相关性(P＞0.05).结果 HT患者外周血中Tfh可能参与桥本甲状腺炎的发生和发展.     

Frequent expression of the natural killer cell receptor KLRG1 in human cord blood T cells: correlation with replicative history

The killer cell lectin-like receptor G1 (KLRG1) belongs to the family of inhibitory C-type lectins that are encoded in the NK gene complex. Similar to other inhibitory NK cell receptors, KLRG1 expression in adult peripheral blood lymphocytes is restricted to NK cells and to antigen-experienced T cells. Umbilical cord blood T cells are thought to represent an homogenous pool of naive T cells. Surprisingly, we identified substantial subsets of CD4 ( approximately 30%) and CD8 ( approximately 20%) alphabeta T cells in cord blood that expressed KLRG1. In contrast to T cells in adult, KLRG1(+) T cells in cord blood exhibited predominantly a naive CCR7(+)CD45RA(+) and CD11a(low) phenotype. After birth, KLRG1 expression in T cells from peripheral blood decreased rapidly to reappear in effector/memory T cells in adults. KLRG1(+) T cells in cord blood expressed a diverse T cell receptor beta (TCRbeta) repertoire and the cells proliferated normally, in contrast to KLRG1(+) T cells from adults. Finally, examination of T cell receptor excision circles (TREC) indicated that KLRG1 expression discriminated between cord blood T cells that differed in their post-thymic expansion rate. Thus, analysis of KLRG1 expression in cord blood revealed an unexpected heterogeneity of human T cells in newborns.