白血病间接免疫荧光法免疫表型分析

目的:研究白血病免疫表型的不同,明确诊断。方法:回顾性分析本院56例白血病患者免疫表型资料。结果:30例急性髓系白血病(AML)均表达髓系抗原,部分伴有淋巴抗原,但其阳性率明显低于急性淋巴细胞白血病(ALL)。7例M3的CD9抗原阳性率为100%,明显高于AML的其它亚型白血病。形态学诊断为ALL的12例白血病中,2例免疫表型为T-ALL,10例B-ALL。4例为杂合性急性白血病,它的诊断标准主要靠免疫分型。在10例慢性粒细胞性白血病(CML)中,4例发生急粒变,且急变期CD34抗原阳性率高于慢性期。结论:免疫表型分析与形态学、细胞化学三者结合为白血病的诊断提供了更可靠、更有价值的依据。     

Ly+-AML和Ly--AML在FAB和WHO分型中的分布及治疗反应

目的: 探讨伴淋系抗原表达的急性髓细胞白血病(Ly -AML)和不伴淋系抗原表达的急性髓细胞白血病(Ly--AML)在FAB和WHO分型中的分布和它们的治疗反应.方法:117例初发的成人急性髓细胞白血病(AML),用常规瑞氏染色及细胞化学染色对骨髓细胞进行FAB分型,采用白血病免疫分型欧洲协作组(EGIL)积分系统,用单克隆抗体标记,经流式细胞仪对骨髓细胞进行免疫分型.其中115例用G显带技术分析染色体核型,按WHO分型标准进行分型.每个病例用标准方案诱导缓解1个疗程后复查血液学缓解情况.结果:117例FAB分型为AML的病例中,免疫学分型为不伴淋系抗原表达的AML(Ly--AML) 89(76.1%)例,伴淋系抗原表达的AML(Ly -AML)28(23.9%)例.WHO分型中,AML伴有重现性染色体异常在Ly -AML和Ly--AML的发生率分别为23.1%和50.5%(P=0.013).用AML的标准诱导治疗方案1疗程后,Ly--AML患者完全缓解率(CR)69.7%,Ly -AML患者CR率34.6%(P=0.001).结论:白血病细胞免疫分型可客观反映细胞来源及分化阶段, 经标准诱导缓解后,Ly--AML患者CR率高于Ly -AML(P=0.001).     

Hoxa家族基因在白血病中的表达及意义

用流式细胞仪对骨髓细胞进行白血病细胞免疫分型,用RT-PCR方法主要检测了急性白血病样品中Hoxa1-13和Meis1基因的表达.结果表明在所有急性白血病患者中CD45和HLA-DR表达阳性,无T系抗原CD3表达,髓系抗原CD33和CD13阳性比例高(76%),B系抗原CD19阳性率为47%,而CD10和CD20阳性率为24%~29%.在急性白血病中Hoxa9和Meis1表达正相关,Hoxa6和Hoxa2表达大多呈现正相关,Hoxa1和Hoxa3在大多数样品中表达负相关.Hoxa3只在CD19阴性CD7阳性的急性白血病样品中表达.结果提示在急性白血病中Hoxa家族基因的表达异常,具有一定的表达相关性,并且免疫表型具有异质性,免疫抗原与Hoxa基因存在着一定的联系.     

急性髓系白血病微小残留病灶检测方法的建立及其应用

目的 依据白血病相关免疫表型的抗原表达规律,建立急性髓系白血病微小残留病灶检测方法.方法 应用多参数流式细胞术对白血病细胞特征性抗原进行检测,所用荧光抗体涵盖了抗原跨系表达、抗原跨阶段表达,并注意抗原过度、过低或缺失表达以及光散射异常等情况,计算白血病细胞占全部检测细胞百分率,即MRD检测量.结果 23例患者抗原不同步表达CD33\CD13\CD15\CD34\CD45的抗体组合应用最多,占56.52％(13/23);CD33\CD13\CD15\CD117\CD45的抗体组合占21.74％(5/23).抗原跨系表达的抗体中CD33\CD13\CD19\CD34\CD45和CD33\CD13\CD7\CD34\CD45比例相对较少,分别为17.39％(4/23)和4.35％(1/23).生存期大于2 a的患者MRD数值明显低于生存期小于2 a的患者,差异具有统计学意义(P<0.01).结论 急性髓系白血病微小残留病灶检测是临床预后判断的重要参考指标.     

正常成人外周血全血培养激活T细胞表达活化抗原

目的：研究正常成人外周血T细胞体外活化表达活化抗原CD69，CD25及HLA－DR的规律与蛋白激酶C（PKC）抑制剂对此的影响。方法：健康志愿者（10名）外周血以佛波醇酯（PDB）＋离子霉素（lon)或植物血凝素（PHA）刺激不同时间后，经双荧光染色后获取有核细胞，以流式细胞仪测定CD3^ T细胞活化抗原CD69，CD25及LHA－DR的表达。结果：无论PDB＋Ion还是PHA均可刺激CD3^ T细胞表达CD25,24h后的表达率分别达57．5％和39．8％，以PDB＋Ion刺激4h，T细胞CD69表达已达90％以上，而此时CD25尚未表达，HLA－DR则在72h后表达明显上调，PKC抑制剂H7能完全抑制DB＋Ion引起的CD69及CD25上调，但仅部分抑制LA－DR的表达。结论：正常成人中血CD3^3 T细胞经多克隆活化剂刺激后CD69表达最快，最高，CD25次之，HLA－DR较慢较低；三者的表达均可被PKC抑制剂H7所抑制，表明PKC在3种活化抗原的表达中起重要作用。     

食管癌组织HLA-I类抗原及相关分子的表达及意义

为探讨HLA抗原及相关分子在食管癌中的表达水平及其与病理学分型的关系，应用了5种HLA抗原及相关分子单抗，采用免疫组织化学方法(ABC法)对江苏省83例食管癌组织石蜡切片进行检测并结合肿瘤的临床病理资料综合分析．得出结果：HLA—B／C、HLA—A、β2m、LMP2、calnexin各分子的下调率分别为12％、25．3％、15．7％、19．3％、20．8％；丢失率分别为29％、33．7％、49．3％、24．1％、41．5％．其中LMP2位点的表达与肿瘤分型相关，随着食管癌分化程度降低，下调率增加．27例病例有淋巴结转移，但统计学分析各分子的表达与转移均无明显相关性．研究表明，食管癌中有明显的HLA—Ⅰ类分子表达下调或缺失．这种改变不利于T细胞依赖性的免疫治疗．     

人类白细胞抗原HLA—DP，DQ，DR在新疆哈萨克族食管癌中的表达及意义

探讨人类白细胞抗原（HLA—DP,DQ,DR）在新疆哈萨克族食管鳞癌发生发展中的表达及其临床意义。采用免疫组织化学技术链霉素-过氧化物酶（S-P）法检测66例新疆哈萨克族食管鳞癌和28例正常食管粘膜组织（癌旁正常组织）中HLA—DP,DQ,DR抗原的表达。新疆哈族食管鳞癌组织中HLA—DP,DQ,DR抗原的阳性表达率为36．4％,高于正常对照组的7．1％（χ^2=8．389,P〈0．05）;HLA—DP,DQ,DR抗原在食管癌中的表达与患者的年龄、性别、肿瘤生长部位、淋巴转移、分化程度无相关性。HLA—DP,DQ,DR的表达与新疆哈族食管鳞癌的发生发展密切相关。     

CpG岛甲基化检测方法

目的：建立CpG岛甲基化测定方法,方法：收集20例急性粒细胞性白血病（AML）,18例多发性骨髓瘤（MM）,14例骨髓异常增生综合征（MDS）,15例慢性粒细胞性白血病（CML）及20 例正常对照组的外周血,分离单个核细胞,提取DNA,应用CpG岛思虑在化特异的方法（MSP－PCR）,测定P15和P16基因甲基化情况,结果：P15和P16基因甲基化在各种白血病的表率率分别为AML 80％和70％,MM72．2％和66．7％,MDS 57．1％和50％,CML0％,结论：P15及P16基因甲基化在AML,MM及MDS中有较高表达,而在CML中不表达。     

饲养环境对C57BL/6J近交系小鼠免疫特性的影响

对普通级和清洁级C5 7BL 6J近交系小鼠的免疫指标CD4 + 、CD8+ 、CD3+ 、CD19+ 进行了测定 ,结果表明 :清洁级与普通级小鼠的CD8+ 、CD4 + CD8+ 指标差异显著 ,并且清洁级小鼠的CD4 + CD8+ 值低于普通级小鼠。为研究清洁级实验动物的特性提供基础数据     

恶性实体瘤患者自体CIK细胞的体外大量扩增后的免疫表型变化

目的:回顾性分析我院2009年1月至2010年5月行细胞因子诱导的杀伤细胞(CIK)治疗的59例实体瘤患者的自体CIK细胞体外扩增后免疫表型的变化,为恶性实体瘤患者开展CIK细胞治疗提供实验依据。方法:采集59例恶性实体瘤患者外周血单个核细胞,培养体系加入IFN-γ、CD3McAb及IL-2三种细胞因子体外诱导,培养10～14 d;用流式细胞仪分别检测CIK细胞培养前后的免疫表型,进行配对t检验。结果:CIK细胞培养后单个核细胞,CD3+细胞,CD3+CD4+细胞,CD3-CD56+细胞,CD3+CD56+细胞均较培养前增加,差异有统计学意义(P<0.05)。CD3+CD8+细胞,虽然较前增加,但差异无统计学意义(P>0.05),CIK细胞中CD3+,CD3+CD4+,CD3+CD8+,CD3-CD56+表型的细胞比率与培养前相比均有所下降,差异有统计学意义(P<0.05),但CD3+CD8+细胞比率与培养前差异无统计学意义(P>0.05)。而CIK细胞的纯度从培养前(9.90±8.96)%增至(46.55±19.25%),差异有统计学意义(P<0.05)。结论:恶性实体瘤患者自体CIK细胞体外扩增后,CIK细胞纯度显著增加。     

Characterization of a common precursor population for dendritic cells

Dendritic cells (DCs) are essential for the establishment of immune responses against pathogens and tumour cells, and thus have great potential as tools for vaccination and cancer immunotherapy trials. Experimental evidence has led to a dual DC differentiation model, which involves the existence of both myeloid- and lymphoid-derived DCs. But this concept has been challenged by recent reports demonstrating that both CD8- and CD8+ DCs, considered in mice as archetypes of myeloid and lymphoid DCs respectively, can be generated from either lymphoid or myeloid progenitors. The issue of DC physiological derivation therefore remains an open question. Here we report the characterization of a DC-committed precursor population, which has the capacity to generate all the DC subpopulations present in mouse lymphoid organs---including CD8- and CD8+ DCs, as well as the B220+ DC subset---but which is devoid of myeloid or lymphoid differentiation potential. These data support an alternative model of DC development, in which there is an independent, common DC differentiation pathway.     

50例急性白血病细胞电镜研究

用电镜和光镜相结合观察50例急性白血病,两者诊断符合率达90%,电镜有助于急性白血病的诊断分型,SEM 下急粒以嵴样型细胞为多,占48～90%;急单中的皱膜型细胞为70～81%;急淋白血病细胞表面特征主要有光滑型和微绒毛型两种.TEM 观察结果和既往作者报道的相似.     

免疫治疗现重要突破,靶向药物创历史进展——2017年癌症治疗热点回眸

 癌症治疗在2017年迎来了一系列新的希望,其中最为重要的是CAR-T治疗的正式登场,两种靶向CD19的CAR-T细胞获得对复发性/难治性B细胞急性淋巴细胞白血病以及大B细胞淋巴癌治疗的批准。同时,免疫检查点抑制剂也获得更多实体瘤适应症的批准,尤其是将MSI-H或dMMR作为生物标志物,作为临床治疗的指导。在靶向治疗方面,一些新的药物获得批准,比如FLT3突变抑制剂Midostaurin与化疗的联合使用,是25年来FDA首次批准的AML新药。此外,在临床研究中也出现一些比较激动人心的结果,癌症疫苗在黑色素瘤的治疗中获得了突破。本文将对这些热点研究结果进行回顾性综述。     

PCR检测白血病患者外周血中EB病毒的DNA

目的：了解白血病患者ＥＢ病毒感染情况。方法：收集２１例急性淋巴细胞白血病、１例慢性淋巴细胞白血病、１５例急性粒细胞白血病、８例慢性粒细胞白血病患者及３２例正常对照组的外周血,分离单个核细胞,提取ＤＮＡ,应用ＰＣＲ方法检测ＥＢ病毒ＤＮＡ。结果：在１例初诊慢性粒细胞白血病病人样本中发现ＥＢ病毒阳性,余均为阴性。结论：白血病患者存在ＥＢ病毒感染情况,但并不普遍。     

Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.     

Formation of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells.

Haematopoietic stem cells are a population of cells capable both of self renewal and of differentiation into a variety of haematopoietic lineages. Enrichment techniques of human haematopoietic stem cells have used the expression of CD34, present on bone marrow progenitor cells. But most CD34+ bone marrow cells are committed to their lineage, and more recent efforts have focused on the precise characterization of the pluripotent subset of CD34+ cells. Here we report the characterization of two distinct subsets of pluripotent stem cells from human fetal bone marrow, a CD34+, HLA-DR+, CD38- subset that can differentiate into all haematopoietic lineages, and a distinct more primitive subset, that is CD34+, HLA-DR-, CD38-, that can differentiate into haematopoietic precursors and stromal cells capable of supporting the differentiation of these precursors. These data represent, to our knowledge, the first identification of a single cell capable of reconstituting the haematopoietic cells and their associated bone marrow microenvironment.     

A novel family of human major histocompatibility complex-related genes not mapping to chromosome 6

Thymocyte antigens CD1 [Thy,gp45,12] are thought to be the human counterparts of mouse thymus leukaemia (TL) antigens. Serological and biochemical analyses indicate that at least three subsets exist, the first of which (HTA 1/T6) was initially identified by the monoclonal antibody NA1/34. Like TL, CD1 are expressed on cortical thymocytes as well as on some lymphoid neoplasias, and resemble in structure major histocompatibility complex (MHC) class I antigens. However HTA 1/T6 is loosely associated with beta 2-microglobulin and is also found linked by a disulphide bridge to CD8(T8). A molecular genetic approach is needed to investigate the CD1 system, to clarify its relationship to TL antigens and to understand its regulation. We report the isolation of complementary DNA (cDNA) clones encoding a CD1 antigen. These clones reveal a novel family of genes which are MHC-related but are neither equivalent to mouse TL antigens nor linked to the MHC.     

High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR

Staphylococcal enterotoxins A-E (refs 1-3), toxic shock toxin (TST-1) (ref. 1), a product of Mycoplasma arthritidis and the Mls antigens provoke dramatic T-cell responses. All are extremely potent polyclonal mitogens stimulating a large proportion of both murine and human CD4+ and CD8+T cells although activity is tightly restricted by major histocompatibility complex (MHC) class II antigens. The murine T-cell response to staphylococcal enterotoxin B (SEB) has recently been shown to involve only those T cells expressing T-cell receptor V beta 3, 8.1, 8.2 and 8.3 domains, a situation which closely mimics the response to Mls antigens. This paper examines the initial events in SEA and SEB T-cell activation and shows that MHC restriction results from a direct high affinity binding by intact SEA and SEB to the same site on MHC class II HLA-DR antigens.