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.     

急性单核细胞白血病病人外周血TCR Vβ T细胞克隆性增殖特点

目的:了解急性单核细胞白血病(ANLL-M5亚型)病人TCR Vβ亚家族T细胞的分布及其克隆性。方法:利用RT-PCR分别扩增9例M5病人外周血单个核细胞的TCR Vβ 24个亚家族基因的CDR3,了解病人各Vβ亚家族的利用情况。阳性的PCR产物进一步经荧光素标记和基因扫描分析产物的CDR3长度,了解T细胞克隆性。结果:9例病人仅存在1-10个Vβ亚家族T细胞。基因扫描分析显示,8例病人外周血中的某些Vβ亚家族出现寡克隆性T细胞,Vβ2寡克隆T细胞发现于6例病人中,2例分别存在Vβ7或Vβ9克隆性T细胞,2例除Vβ2外,还分别出现Vβ7或Vβ21的寡克隆T细胞。结论:结果提示M5病人外周血Vβ亚家族T细胞的倾斜性分布特点,并存在克隆性增殖的T细胞,这可能是机体T细胞受白血病细胞相关抗原的刺激作用而引起机体产生相应的特异性免疫反应,并以Vβ2的克隆性增殖T细胞为主,提供了明显的趋向性,它可能与M5细胞相关抗原有关。     

急性单核细胞白血病病人外周血TCR Vα基因谱系和T细胞克隆性增殖特点

目的：了解急性单核细胞白血病（AML-M5）病人TCR Vα29个亚家族的分布及其克隆性。方法: 利用RT-PCR分别扩增8例M5病人外周血单核细胞的TCR Vα29个亚家族基因的CDR3。阳性的PCR产物进一步经荧光素标记和基因扫描分析产物的CDR3长度，了解T细胞克隆性。9例健康人作为对照。结果: RT-PCR分析显示正常人表达绝大多数Vα亚家族基因，而8例M5病人外周血仅可以检测到1-10个Vα亚家族基因，出现频率最高的是Vα3（6/8例，75%），其次为Vα12（5/8例，62.5%），有15个Vα亚家族表达缺失（Vα1、4、5、7、9、14-18、20、21、26、28和Vα29）。基因扫描分析显示：8例AML-M5病人中有6例存在克隆性增殖T细胞，其中，以Vα12亚家族出现克隆性增殖T细胞频率（3/5例）最高，有2例仅存在单一的克隆性增殖Vα3亚家族T细胞。正常人外周血各Vα亚家族T细胞主要呈多克隆性。结论: M5病人外周血Vα亚家族T细胞存在明显的选择性选用及克隆性增殖特点，这可能是机体T细胞受M5细胞刺激而引起机体产生相应的特异性免疫反应，同时T细胞TCR Vα亚家族分布及克隆性增殖情况具有个体特异性。     

分化早期小儿急性白血病细胞免疫球蛋白和T细胞受体基因结构的变化

从29例免疫分型的小儿急性白血病中选出髓过氧化物酶染色阴性和不与细胞系相关或成熟细胞系相关的单克隆抗体反应的6例白血病细胞,进一步作免疫球蛋白(包括重链和κ轻链)和T细胞受体(包括δ、γ、β)基因结构的分析。所有6例均有IgH基因的重排,提示这些白血病细胞是β细胞来源。两例CD_(10)阴性和白病细胞.κ链基因没有重排,其中一例的TCRδ、γ、β基因也都处于胚系,另一例的TCRβ处于胚系。在CD_(10)阳性白血病细胞中,其中两例κ链基因丢失,TCR基因结构都有不同程度的变化(重排或丢失)。这些结果可能提示在CD_(10)阳性白血病细胞中能产生功能性IgH的重排,并可导致IgL和TCR基因结构的变化。     

T-ALL及T细胞株相关TCR Vβ基因谱系和克隆性分析

目的了解T细胞-急性淋巴细胞白血病(T-ALL)患者外周血中的T细胞及T细胞株的TCR Vβ基因谱系及其克隆性增殖情况。方法利用RT-PCR方法扩增6个不同的T细胞株和6例初发未治T-ALL病人外周血单个核细胞中24个TCR Vβ基因的互补决定区3(CDR3)，PCR产物进一步经荧光标记和基因扫描分析CDR3长度而确定T细胞的克隆性，部分T细胞株的单克隆PCR产物进一步进行序列分析。结果与正常人外周血表达全部24个Vβ亚家族不同，6例T-ALL病人分别表达5～12个Vβ亚家族。6例病人均存在1个或多个Vβ亚家族的寡克隆或双克隆增殖T细胞，另外，还有一些Vβ亚家族多克隆模式发生改变，呈现寡克隆性增殖的趋势。T细胞株多显示为表达一个Vβ亚家族的单克隆T细胞，不同T细胞株的CDR3长度和序列不尽相同。结论T-ALL患者外周血T细胞的TCR Vβ谱系出现限制性改变，均可检测到克隆性增殖T细胞，尚需进一步鉴定其性质(肿瘤性或抗原特异性增殖)，对于检测微小残留病变和设计抗白血病独特型疫苗均有一定的意义。     

小儿急性淋巴细胞白血病的免疫分型和抗原受体基因分析

小儿急性淋巴细胞白血病的免疫分型和抗原受体基因分析刘筱萍费洪宝汪俊华梁欣荃近年来，用分子生物学技术在基因水平检测免疫球蛋白和Ｔ细胞受体基因的重排，从本质上区分Ｂ系和Ｔ系，与免疫分型结合起来分析已成为一种对急性淋巴细胞白血病（急淋）分型的较理想手段。１...     

T 细胞受体δ、γ基因在小儿急性白血病中的变化

本文在细胞形态,表面抗原分析的基础上,对20例小儿急性白血症细胞进行TCRδ和γ基因结构的分析。11例属分化早期的白血病中有9例发生TCRδ基因的重排和缺失,其中5例也有TCRγ基因的重排。在5例属非淋巴细胞系的白血病中有3例发生TCRδ基因的重排.3例粒细胞性白血病细胞的TCR基因(δ和γ)均为胚系。有一例细胞表型仅有CD_2表达的白血病细胞,其TCR 基因(δ和γ)也为胚系。结果还显示:TCRδ基因结构发生变化的白血病细胞均有CD_(10)抗原表达。研究提示:TCRδ基因的重排不显示严格的细胞特异性,但是TCR 基因的变化与淋巴细胞系白血病细胞某些表面抗原的表达存在某种联系。     

microRNA-204 inhibits cell proliferation in T-cell acute lymphoblastic leukemia by down-regulating SOX4

Background: MicroRNAs (miRNAs) are a group of small non-coding RNAs that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. The aim of this study was to explore the effect of miR-204 on cell proliferation migration and invasion in T-cell acute lymphoblastic leukaemia (T-ALL). Method: miR-204 expression was determined in bone marrow samples from 32 leukemia patients and 32 healthy controls by quantitative real-time PCR (qRT-PCR). The effect of miR-204 on cell proliferation was evaluated by CCK8 assay, cell migration and invasion were evaluated by transwell migration and invasion assays, In addition, the regulation of SOX4 by miR-204 was evaluated by luciferase reporter assay and western blot. Results: our results revealed that miR-204 was low expressed in T-ALL. Cell proliferation assay showed that the cell proliferation ability was inhibited by miR-204 mimics. Moreover, migration and invasion assay suggested that overexpression of miR-204 could significantly suppressed the migration and invasion ability of T-ALL cells. Luciferase reporter assay confirmed that miR-204 directly bound to the 3’ untranslated region of SOX4, and western blot suggested that miR-204 inhibited the expression of SOX4 at the protein levels. Conclusions: Our findings indicated that miR-204 negatively regulates SOX4 and inhibited proliferation, migration and invasion of T-ALL cell lines. Thus, miR-204 might represent a potential therapeutic target for T-ALL intervention.     

111例急性T淋巴细胞白血病免疫表型分析

选用一组抗人白细胞表面分化抗原单克隆抗体和基因探针,依靠间接免疫荧光技术、Ap-AAP桥联酶标免疫组化染色和分子杂交手段.对111例急性T淋巴细胞白血病(T-ALL)进行了免疫分型研究。本研究所观察到的T-ALL细胞表型绝大多数反映着正常胸腺细胞分化发育阶段或某些少见类型细胞的表型特征,亦有少数病例则无正常淋巴细胞相对应的表型,表明T-ALL细胞的高度异质性。对T-ALL表型的研究为今后深入探讨胸腺内淋巴细胞的分化成熟以及T-ALL发生学提供了良好的研究模式。     

Demonstration of clonality in T-cell lymphoma using an anti-T-cell receptor variable region antibody panel

Frozen sections from 35 T-cell lymphomas were stained with the Diversi-T alpha beta T-Cell Receptor panel which includes seven antibodies to T-cell receptor variable region gene products. In five cases a monoclonal population of T-cells could be demonstrated (one case V beta 5+, three cases V beta 8+ and one case V beta 6+) and in a further case a biclonal population (V beta 5+ and V beta 8+). We conclude that this antibody panel is of limited usefulness for the demonstration of clonality in T-cell lymphoma.     

Expression and significance of CD47, PD1 and PDL1 in T-cell acute lymphoblastic lymphoma/leukemia

Although dose intensi?cation strategies achieve a favorable prognosis for pediatric patients of T-lmphoblastic lymphoma/leukemia (T-LBL/ALL), numerous side effects have been followed. Molecular targeted therapies will be needed to optimize the current treatment strategy for T-LBL/ALL. The aim of this study was to analyse expression and significance of CD47, PD1 and PDL1?in. T-LBL/ALL. We performed immunohistochemistry staining and real time fluorescence quantitative PCR (qRT-PCR) on FFPE tissues. Immunohistochemistry results showed that the high expression rate of CD47 protein was 46.4% (26/56) and the positive expression rate of PDL1 protein was 37.5% (21/56). PD1 expression was observed in tumor infiltrating lymphocytes in approximately 20% of T-LBL/ALL patients, but not expressed on tumor cells of T-LBL/ALL. And the results of qRT-PCR showed that the relative expression levels of CD47, PDL1 and PD1 mRNA in 56 cases of T LBL/ALL were significantly higher than those in control group (6.915 vs 4.050, 12.255 vs 2.575, 37.990 vs 3.615), and the differences were all statistically significant (p all <0.05). Univariate analysis showed that age, CD47 protein, CD47 mRNA,PDL1 protein and PDL1 mRNA expression were closely correlated with prognosis (P all <0.05). We found that the overall one-year survival rates of patients with a high expression (≥M) of CD47 and PDL1 mRNA were higher than in patients with low expression (<M). However, the overall one-year survival rate of patients with a high expression (≥M) of CD47 and PDL1 protein were lower than in patients with low expression (<M). And patients with ≤25 years old had a worse prognosis than with >25 years old. Multivariate Cox regression analysis showed that the high expression of CD47 and PDL1 protein were independent prognostic factors (both p?<?0.05). In a word, PD1/PDL1 and CD47 may be involved in the disease progression and prognosis of T-LBL/ALL, and detection and targeting of CD47 and PD1/PDL1 may provide a rational basis to for treatment of T-LBL/ALL.     

慢性粒细胞白血病相关的TCR Vβ T细胞的诱导及其限制性和克隆性分析

目的:了解体外诱导CML细胞相关TCRVβ亚家族T细胞克隆性增殖及其杀伤性的情况。方法:采用混合淋巴细胞/白血病细胞培养法(MLTC)体外将CML细胞、K562细胞和bcr-abl多肽与供者外周血单个核细胞混合培养和扩增,利用RT-PCR-基因扫描技术分析培养后T细胞的TCRVβ谱系的限制性利用和克隆性增殖情况,并经LDH法分析诱导增殖T细胞的杀伤性。结果:经bcr-abl多肽、CML细胞和K562细胞诱导扩增1-2周后,供者外周血T细胞表达10-13个Vβ亚家族,在Vβ16和Vβ21出现克隆性增殖T细胞,在Vβ5和Vβ13出现寡克隆生长趋势T细胞。诱导扩增后的T细胞对CML和K562细胞具有特异性杀伤作用。结论:利用CML细胞、K562细胞和bcr-abl多肽可在体外诱导出CML细胞特异性CTL,该CTL可能为优势表达的Vβ亚家族克隆性T细胞。     

急性非淋巴细胞白血病患者NK细胞亚群及其NCR受体测定

目的： 探讨初发的急性非淋巴细胞白血病患者NK细胞亚群分布及自然细胞毒受体（NCR）的水平。方法：应用流式细胞仪技术测定26例初发急性非淋巴细胞白血病患者外周血NK细胞、NK细胞亚群、自然细胞毒受体。结果： 初发急性非淋巴细胞白血病患者外周血NK细胞明显低于对照组（P<0.01），CD56bright及CD56dimNK细胞明显低于正常对照组（P<0.01），NKp30、NKp44、NKp46在CD56+CD3-细胞中的表达均低于正常对照组（P<0.05）。结论： 急性非淋巴细胞白血病发病可能与NK细胞及亚群减低，自然细胞毒受体表达降低有关。