B淋巴增殖性疾病的SOX11、cyclin D1、cyclin D2和cyclin D3的表达研究

目的 探讨各B淋巴增殖性疾病(B-LPD)中SOX11、cyclin D1、cyclin D2和cyclin D3表达的差异和相关性,以及与慢性淋巴细胞白血病(CLL)患者临床特征的关系.方法 采用实时定量逆转录PCR(qRT-PCR)技术检测154例B-LPD患者与12例健康对照SOX11、cyclin D1、cyc...     

六亚甲基二乙酰胺对急性白血病细胞株HL-60和U937分化、凋亡的影响及其机制

目的　研究六亚甲基二乙酰胺 (HMBA)体外诱导HL 6 0和U937细胞分化、凋亡的作用及其机制。方法　流式细胞仪检测细胞分化抗原CD1 1b、CD1 4,细胞凋亡标记Annexin Ⅴ以及进行细胞周期分布和细胞内cyclinD、cyclinE、p2 7抗原的分析 ,RT PCR检测c myc、Rb、Bcl 2基因mRNA的表达。结果　HL 6 0、U937细胞经HMBA处理 72h后CD1 1b表达显著增高 ,高剂量HMBA促使Annexin Ⅴ表达增加。HMBA阻滞HL 6 0、U937细胞于G0 G1 期 ,并使该两种细胞内cyclinE表达显著下降 ,cyclinD、p2 7表达显著增高 ,呈剂量依赖关系 ;HMBA可使HL 6 0、U937细胞c myc、Bcl 2mRNA表达下调 ,而RbmRNA在HL 6 0细胞表达上调 ,在U937细胞则无显著改变。结论　HMBA能诱导HL 6 0、U937细胞出现明显的分化 ,高剂量的HMBA有促使HL 6 0、U937细胞凋亡的倾向 ,其机制可能是通过影响细胞周期调控分子以及有关的增殖分化相关基因的表达 ,从而抑制细胞增殖 ,促进细胞分化。     

磷酸二酯酶7B在套细胞淋巴瘤患者中的表达及临床意义

为了研究磷酸二酯酶(PDE)7B在套细胞淋巴瘤(MCL)患者中的表达及临床意义,应用实时定量PCR(QPCR)技术检测20例初治的有骨髓侵犯MCL患者骨髓和20例正常人外周血单个核细胞中pde7b的表达水平,并分析其与MCL临床预后指标的关系。结果表明:20例MCL患者pde7b表达水平中位数为8.7×10-4(4×10-5-6.9×10-3),明显高于正常人群0.5×10-4(0.18×10-4-1.7×10-4)(p=0.001);pde7b与性别、年龄、白细胞计数、乳酸脱氢酶水平、CD38表达和免疫球蛋白重链基因突变均无明显相关,而与细胞遗传学、β2微球蛋白、ZAP-70表达有明显相关。结论:pde7b基因在MCL中表达明显高于正常人群,并与细胞遗传学关系密切,可能成为MCL新的预后指标,具有重要的临床意义。     

急性白血病患者周期蛋白D1mRNA表达及其临床意义

目的　检测急性白血病患者周期蛋白 (cyclin)D1mRNA的表达 ,并探讨其与临床的关系。方法　采用逆转录 聚合酶链反应 (RT PCR)技术检测 6 5例急性白血病患者及 5名正常人骨髓细胞cyclinD1mRNA。结果　正常骨髓未检测到cyclinD1mRNA表达 ;急性白血病患者阳性率 2 6 2 % ,急性淋巴细胞白血病与急性非淋巴细胞白血病之间差异无显著性 ;复发组患者的cyclinD1mRNA阳性率 (6 3 6 % )明显高于初治组 (2 1 4% )及缓解组 (8 3% )。结论　急性白血病患者中有cyclinD1mRNA过度表达的情况 ,且与临床复发关系较为密切。     

microRNA-155及microRNA-146a在慢性淋巴增殖性疾病中的表达及临床意义

目的 观察microRNA-155及microRNA-146a在慢性淋巴细胞白血病(CLL)、套区细胞淋巴瘤(MCL)及脾脏边缘区淋巴瘤(SMZL)患者CD19+B淋巴细胞中的表达,并分析其临床意义.方法 以53例CLL、13例MCL、9例SMZL患者和12名正常人为研究对象.取外周血(78份)或骨髓(9份).常规分离单个核细胞,并行CD19磁珠分选.提取CD19+细胞总RNA,应用TaqMan探针法检测microRNA-155及microRNA-146a的表达,结合患者临床资料进行统计学分析.结果 ①micro RNA-155在CLL患者中的表达(4.49±0.83)显著高于MCL(3.83 ±0.45)及SMZL(3.80±0.61)(P＜0.05);②SMZL患者中microRNA-146a的表达(3.81±0.59)显著高于CLL(2.58±0.90)及MCL(2.21±0.88)(P＜0.01);③CLL IgVH未突变组患者microRNA-155的表达显著高于突变组患者(P=0.012);④CLL患者不同细胞遗传学分组间microRNA-155及microRNA-146a的表达差异无统计学意义(P＞0.05).结论 ①不同慢性淋巴增殖性疾病(LPD)之间microRNA-155及microRNA-146a的表达存在差异;②提示microRNA可能与慢性LPD的发病甚至预后相关.     

转录因子PAX5在儿童急性白血病细胞中的表达

为了观察了解儿童急性白血病细胞中转录因子PAX5的表达特性，采用实时定量RT—PCR方法测定了6个血液肿瘤细胞株以及6例正常儿童、58例初发和4例复发急性白血病儿童（其中包括39例B—ALL，10例T—ALL和13例AML）骨髓细胞中PAX5和CD19mRNA的表达水平。结果表明：在Namalwa（B细胞系）细胞株中，PAX5和CD19mRNA相对表达量分别为2．35％和2．52％；而在T-和髓系细胞株中几乎不表达。在临床样本中B—ALL组比T—ALL组和AML组的PAX5mRNA表达高（P=0．029和P=0．013）。T—ALL组和AML组的PAX5mRNA表达水平均低于正常对照组，而T—ALL组与AML组之间的表达差异无显著意义。在B—ALL患儿中，PAX5mRNA表达的个体差异很大。此外还发现，初发治疗前组和复发组的B—ALL患儿PAX5mRNA表达高于化疗完全缓解组（P=0．011和P=0．006）。由于在CD19基因的启动子上有B细胞特异性激活蛋白的结合位点，研究发现在B-ALL中PAX5表达水平与同样用实时定量RT—PCR检测的CD19表达水平之间存在明显的相关性。结论：运用实时定量RT—PCR方法能快速准确地在B—ALL的临床标本中检测和定量PAX5基因的表达。研究中发现部分B—ALL中PAX5mRNA表达明显升高，因此它可将其作为进一步研究B—ALL发病机理的另一个切入点。     

Ileocolonic lymphomas: a series of 16 cases

BACKGROUND: Colonoscopic and clinical differences between primary ileocolonic mucosa-associated lymphoid tissue (MALT) lymphoma and mantle cell lymphoma (MCL) have not been defined. METHODS: We reviewed colonoscopic and clinical features in eight patients with primary MALT lymphoma and eight patients with MCL in the terminal ileum and/or colorectum. All cases were examined for CD5 and/or cyclin D1 expression. RESULTS: Endoscopic features of MALT lymphoma were characterized as protrusions that were covered with normal-appearing mucosa with or without ulceration. The gross appearances of MALT lymphomas were categorized as solitary (4 patients), multiple (3 patients), and multiple lymphomatous polyposis (MLP) (1 patient). The gross features of MCL at endoscopy were categorized as multiple protrusions (2 patients), and MLP (6 patients). The clinical stages of patients with MCL were more advanced than in patients with MALT lymphoma. CONCLUSIONS: Solitary or multiple protrusions at an early clinical stage is the most common presentation pattern of patients with MALT lymphoma, but an MLP appearance at an early stage is also possible. On the other hand, MLP appearance with an advanced clinical stage is the main presentation pattern in patients with MCL, although multiple protrusions with an early clinical stage is also possible. Histological and immunohistochemical investigation including that of cyclin D1 and CD5 expression is essential to make the final diagnosis.     

用流式细胞术从B淋巴增殖性疾病中鉴别脾边缘带淋巴瘤

脾边缘带淋巴瘤（Splenicmarginalzonelymphoma,SMZL）是一种相对少见的原发于脾脏的惰性B淋巴增殖性疾病,表现为外周血淋巴细胞计数和／或比例增高、脾大,而外周浅表淋巴结往往不大,需要应用诊断性脾切除病理检查得以确诊,但患者多数不能接受,早期诊断困难。本研究探索以流式细胞术（flowcytometry,FCM）为主的诊断途径的可行性。选取6例疑诊SMZL患者为研究对象,同时以10名骨髓健康供者及确诊的10例慢性淋巴细胞白血病（CLL）、3例毛细胞白血病（HCL）、3例淋巴浆细胞淋巴瘤／华氏巨球蛋白血症（LPL／wM）初诊患者为对照。对所有患者及对照组进行骨髓FCM免疫分型,所选抗体组合包括CD45、CD5、CD10、CD19、CD20、CD22、CD23、CD25、CD103、CD11c、CD123、κ、λ、CyclinD1等,同时结合骨髓细胞形态学检查。结果表明：6例疑诊sMzL患者表现为淋巴细胞增高和脾大。因外周淋巴结无肿大6例患者均未行淋巴结活检,仅1例患者行病理诊断性脾切除。经骨髓FCM免疫表型分析,除健康供者外均可发现骨髓中存在不同程度的异常成熟B细胞克隆,并通过CD5、CD10表达与否结合其他表型进一步与其他B细胞肿瘤鉴别。结果6例SMZL患者均CDl9＋、CD20＋,而CD10-,4例患者CD5-,2例CD5＋,而CD23、CD38、ZAP-70、CD11c、CD103、CD123、CyclinD1均不表达。骨髓细胞形态学检查可见有短绒毛的异常淋巴细胞,结合临床特征将6例患者诊断为SMZL。1例患者因合并脾功能亢进需要行脾切除,术后病理证实该病。10例CLL主要表达CD5、CD23之外;在3例HCL除表达CD11c、CD103、CD123,形态学上更有典型的”毛”;3例LPL／WM具有轻链限制性表达、IgM显著升高、浆样淋巴细胞增多的特点。结论：FCM免疫表型分析结合骨髓形态学可作为临床诊断SMZL有力的工具。     

FP248基因表达检测在白血病和肿瘤诊断中的意义

目的 用real-time PCR和RT-PCR检测FP 248 mRNA表达,研究其在白血病和肿瘤诊断中的意义.方法 用real-timePCR和RT-PCR检测肿瘤细胞株(SGC-7901、A549、95D、SW480、U937、PC3)和白血病(ALL、CML、AML)患者骨髓或外周血以及胃癌等消化道肿瘤患者癌组织中FP248 mRNA的表达水平;用RT-PCR技术检测消化道肿瘤患者外周血单个核细胞(PB-MC)FP 248 mRNA的表达.结果 SGC-7901细胞株FP 248 mRNA强阳性,A549和95D细胞弱阳性,其他细胞株阴性;ALL、CML、AML患者骨髓及PBMC FP 248表达上调;胃癌、直肠癌、结肠癌患者癌组织中FP 248 mRnNA的表达高于癌旁组织,食道癌组织为阴性;胃癌、结肠癌和直肠癌患者PBMC未检测到FP 248 mRNA.结论 FP 248的表达可能在白血病和消化道肿瘤的发生发展中起重要作用;检测该基因的表达可能有助于白血病和消化道肿瘤的诊断.     

Measurement of CD83 mRNA by real-time polymerase chain reaction to determine removal of dendritic cells by leucoreduction of whole blood

Information is lacking regarding efficiency of removal of circulating dendritic cells (DCs) by leucoreduction (LR) of blood. This is important since DCs may play a role in transporting abnormal prion, the likely infectious agent of variant Creutzfeldt-Jakob disease. In this study, we report development of a real-time polymerase chain reaction (RT-PCR) assay to quantify residual DCs in LR whole blood via measurement of selected messenger RNA (mRNA) markers. Taqman-based RT-PCR assays were set up for CD83 as a marker of mature DCs, and CD1c, CD11c, CD303 and CD304 as markers for plasmacytoid and myeloid DCs along with the pan-leucocyte marker CD45. We then assayed 46 paired pre-/post-LR whole blood samples and determined the log(10) reduction of their CD83 and CD45 mRNA. Our data indicate that RT-PCR can be used to detect suitably low CD83 mRNA levels. We measured a median log(10) reduction for CD83 mRNA of 4.5 [standard error of the mean (SEM) 0.07] and 4.1 (SEM 0.10) for CD45 mRNA. These reductions are comparable to cell removal, where flow cytometry indicated a reduction in total white cell counts of 4.3 log(10) (SEM 0.09). Our other group of markers were reduced to their detection limits, CD1c (3.9 log(10), SEM 0.3), CD11c (5.0 log(10), SEM 0), CD303 (3 log(10), SEM 0.1), CD304 (4.0 log(10), SEM 0) which are all higher than the minimum specifications for LR products. In conclusion, we successfully developed an RT-PCR assay to quantify suitably low numbers of DC cells. We show for the first time that DCs are effectively removed using a standard whole blood filter.     

骨肉瘤中D类周期素的mRNA表达

目的 探讨骨肉瘤中D类三种周期素——D1、D2及D3 mRNA的表达。方法运用逆转录聚合酶链反应(RT-PCR)及半定量RT-PCR方法检测正常软组织和骨肉瘤中3种周期素D mRNA表达。结果正常软组织中检测不到周期素D1 mRNA的表达，而骨肉瘤组织中均可检测到，但多数表达较弱；正常软组织及骨肉瘤组织中均存在周期素D2、周期素D3 mRNA的表达，骨肉瘤中周期素D2、周期素D3的mRNA表达均明显高于软组织中的表达。结论周期素D2、D3可能在骨肉瘤失控的增生中起主要作用。     

Residual subset population analysis in WBC-reduced blood components using real-time PCR quantitation of specific mRNA

BACKGROUND: Implementation of WBC reduction of the blood supply increases the importance of measurement of residual WBC subtypes responsible for immunologic and infectious complications of transfusion. STUDY DESIGN AND METHODS: Real-time RT-PCR assays were developed to detect mRNA encoding lineage-specific WBC markers. Primers and fluorescent probes were designed for CD45 (pan-WBC), CD3 (T-lymphocyte), CD19 (B-lymphocyte), CD14 (monocyte), and CD66 (granulocyte), and the specificity was assessed by comparison with flow cytometric analysis of enriched cell populations. WBC subsets were examined in WBC-reduced whole blood prepared with filters (WBF2, Pall; and RZ2000, Baxter) and in platelet concentrates prepared with other filters (Autostop, Pall; and PLX-5, Baxter) and apheresis (COBE Spectra LRS, Gambro). RESULTS: All real-time RT-PCR assays were linear over >5 log concentration range, allowing pre-WBC-reduction and post-WBC-reduction comparisons. Sensitivity limits ranged from 10 cells per mL (CD45) to 200 cells per mL (CD19). Assay specificity was confirmed by the close correlation of real-time RT-PCR and immunophenotyping results by flow cytometry. For all subsets, >3.8 log and >3.1 log reduction was obtained during WBC reduction of whole blood and platelets, respectively. CONCLUSION: Real-time RT-PCR assays are suitable for analysis of subset removal during WBC reduction. There was no significant difference between the two whole-blood filters or between platelet filtration and apheresis in the removal of any WBC subset.