恶性血液病细胞株人端粒重复序列结合因子基因突变情况的研究

目的　检测 1 1个端粒酶阳性的恶性血液病细胞株中人端粒重复序列结合因子 1(TERF1 )基因的突变情况并探讨其意义。方法　用生物医学软件预测TERF1基因组结构并用PCR和测序的方法证实 ,确定TERF1全长基因组结构。采用端粒重复序列扩增 (TRAP) 酶联免疫吸附实验 (ELISA)检测细胞株的端粒酶活性。以PCR产物直接测序的方法检测髓系白血病细胞株K5 6 2、HL 6 0、U937、NB4、THP 1、HEL、Dami,淋巴细胞白血病细胞株 6T CEM、Jurkat和Raji,以及骨髓增生异常综合征 (MDS RAEB)细胞株MUTZ 1中TERF1外显子的突变情况 ,以正常人骨髓单个核细胞作对照。结果　TERF1基因组全长 38.6kb ,由 1 0个外显子和 9个内含子构成 ,外显子和内含子的接合边界获确认。 1 1种恶性血液病细胞株均呈端粒酶阳性。在所检测的细胞株中未发现外显子存在有意义的突变 ,但在外显子周边的部分内含子中发现 4个位点有变异 ,其中MUTZ 1的变异有别于白血病细胞株 ,未发现髓系和淋巴细胞系细胞株之间的变异情况有差异。结论　TERF1外显子突变可能并不是恶性血液病细胞中TERF1功能异常的主要原因     

恶性血液病患者血浆血管内皮生长因子检测及其临床意义

血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)是主要的促血管新生因子 ,国内有关恶性血液病VEGF表达的研究尚少。我们对恶性血液病患者及正常人血浆VEGF进行了观察 ,以期探讨其在恶性血液病中的表达情况及意义。对象和方法1　研究对象　病例 :2 0 0 1年 8月～ 2 0 0 2年 2月我院血液科收治的恶性血液病患者 4 2例 ,男 2 4例 ,女 18例 ,年龄 13～ 80岁 ,中位年龄 5 5岁。其中急性髓系白血病 10例 ,急性淋巴细胞白血病 2例 ,慢性粒细胞白血病 3例 ,慢性淋巴细胞白血病 2例 ,骨髓增生异常综合征 4例 ,非霍奇金淋巴瘤 16例 ,…     

人类端粒酶逆转录酶基因反义核苷酸对白血病细胞端粒酶活性的影响

为了探讨人类端粒酶逆转录酶（hTERT)基因反义寡核苷酸（ASODN）对原代培养的急性髓性白血病（AML）和慢性髓性白血病（CML）细胞端粒酶活性的影响。采用间接免疫荧光标记方法，通过流式细胞术检测hTERT基因ASODN作用于AML和CML细胞后hTERT蛋白表达含量的变化。采用端粒酶聚合酶链反应-酶子弟免疫测定（PCR-ELISA0法检测白血病细胞端粒酶活性的改变。结果显示：hTERT ASODN作用于AML和CML细胞24，48和72小时后，hTERT蛋白表达水平不断降低；同时，AML和CML细胞端粒酶活性均逐渐受到抑制。结论：hTERT基因ASODN可以通过特异性地抑制AML和CML细胞hTERT蛋白的表达，抑制AML和CML细胞端粒酶的活性。     

PAX5/BSAP在儿童急性白血病细胞和血液肿瘤细胞株中的表达研究

目的了解儿童急性白血病细胞和血液肿瘤细胞株中转录因子PAXS／BSAP的表达特性。方法采用实时定量RT-PCR方法，检测了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骨髓细胞中PAX5和CD19mRNA的表达水平。采用Western Blotting分析了6个血液肿瘤细胞株和4个临床样本中BSAP表达水平。采用RNAi（RNA干扰）技术特异性阻断NAMALWA细胞株（B细胞系）中PAX5的表达。结果在NAMALWA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。同时运用Western Blotting在B系细胞中检测到-52KD的BSAP条带，但在T-和髓系细胞中为阴性。在临床样本中B-ALL（急性B淋巴细胞白血病）组比T-ALL（急性T淋巴细胞白血病）组和AML（急性髓细胞性白血病）组的PAX-5 mRNA表达高（P=0．029和P=0．013）。在B-ALL患儿中，PAX-5 mRNA表达的个体差异很大。RNAi技术抑制NAMALWA细胞株中PAX-5基因表达达35％，但不影响细胞增殖效率（P〈0．05）。结论部分B-ALL中PAX-5 mRNA表达明显升高，可作为进一步研究B-ALL发病机理的另一个切入点。RNAi技术可进一步用于B系血液恶性肿瘤细胞的观察研究。     

CD34抗原在急性双表型白血病的临床研究

本研究旨在探讨白细胞CD34抗原在急性双表型白血病（biphenotypic acute leukemia,BAL）的表达情况,以明确其与BAL预后的关系.应用流式细胞仪检测白血病相关抗原,所用单克隆抗体B淋巴细胞系列为CD10,CD19和CD34;T淋巴细胞系列为CD2,CD3和CD5;髓系为MPO,CD13和CD33,最终对结果进行分析.结果表明：216例急性白血病患者中9例（4.2％）被诊断为急性双表型白血病,其中髓系和B淋巴细胞系共同表达6例（66.7％）,髓系和T淋巴细胞系共同表达3例（33.3％）.4例CD34表达阳性（44.4％）与急性髓系白血病和急性淋巴细胞白血病相比,双表型急性白血病患者表现出显着较高的CD34抗原阳性表达率（P＜0.05）.结论：与急性髓细胞性白血病或急性淋巴细胞白血病相比,急性双表型白血病患者的预后较差,CD34阳性表达可能与急性双表型白血病的治疗效果呈负相关.     

恶性血液病细胞端粒酶活性检测及其临床意义

目的研究恶性血液病的端粒酶活性表达情况,探讨端粒酶活性检测对恶性血液病的临床意义。方法选取各类恶性血液病病例87例,以良性血液病和骨髓形态学正常的非血液病患者作为对照,采用改良TRAP-银染法及亚利恩凝胶成像系统检测其骨髓标本内的端粒酶活性水平并随访。结果良性血液病时端粒酶阴性或低水平表达;慢性白血病慢性期、骨髓增生异常综合征(MDS)、多发性骨髓瘤(MM)骨髓端粒酶活性轻度或中度升高,急性白血病和慢性白血病急变期端粒酶活性显著升高,完全缓解后活性水平下降,复发时端粒酶活性又升高。同时端粒酶活性水平与预后相关,活性高者往往预后较差。结论端粒酶活化与肿瘤的恶性转化密切相关,可作为恶性血液病恶性克隆增殖的分子标志,有希望成为监测微量残留白血病的一个新指标。     

恶性血液病细胞端粒酶活性检测及其临床意义

目的　研究恶性血液病的端粒酶活性表达情况,探讨端粒酶活性检测对恶性血液病的临床意义。方 法　选取各类恶性血液病病例87例,以良性血液病和骨髓形态学正常的非血液病患者作为对照,采用改良 TRAP 银染法及亚利恩凝胶成像系统检测其骨髓标本内的端粒酶活性水平并随访。结果　良性血液病时端粒 酶阴性或低水平表达;慢性白血病慢性期、骨髓增生异常综合征(MDS)、多发性骨髓瘤(MM)骨髓端粒酶活性轻 度或中度升高,急性白血病和慢性白血病急变期端粒酶活性显著升高,完全缓解后活性水平下降,复发时端粒酶 活性又升高。同时端粒酶活性水平与预后相关,活性高者往往预后较差。结论　端粒酶活化与肿瘤的恶性转化 密切相关,可作为恶性血液病恶性克隆增殖的分子标志,有希望成为监测微量残留白血病的一个新指标。     

survivin基因在血液肿瘤细胞株的表达

survivin基因是近年来被发现的凋亡蛋白抑制因子家族(IAP)成员之一,其结构简单,组织分布具有明显的特征。我们研究了其在不同类型血液肿瘤细胞株中的表达,现报道如下。对象和方法1　研究对象　将巨核细胞白血病细胞株Dami、Burkitt’s淋巴瘤细胞株Raji、髓系白血病细胞株HL 6 0、组织细胞淋巴瘤细胞株U937、红白血病细胞株HEL、T细胞白血病细胞株6T CEM、慢性髓细胞白血病细胞株K5 6 2、T细胞淋巴瘤细胞株Jurkat、急性早幼粒细胞白血病细胞株NB4、骨髓增生异常综合征RAEB t细胞株MUTZ 1(各细胞株均为我院保种)置于RPMI 16 4 0…     

人脐血造血干/祖细胞体外定向诱导分化过程中端粒酶的表达

探讨脐血造血干/祖细胞体外诱导分化过程中端粒酶的表达,为造血干细胞产品的临床应用提供一个监测细胞增殖潜能和安全性的指标。用源自人脐血的CD34~+细胞及不同细胞因子组合(SCF+IL-3+IL-6+G-CSF,SCF+IL-3+IL-6+EPO)在体外进行诱导分化;用TRAP聚丙烯酰胺凝胶电泳法、TRAP-ELISA法检测CD34~+细胞及诱导分化细胞的端粒酶活性;用Western印迹法在蛋白水平检测端粒酶催化亚基hTERT的表达,用RT-PCR在细胞转录水平检测端粒酶催化亚基hTERT mRNA的表达。结果显示,在体外诱导分化14-21天为细胞生长峰值,细胞总数可增加(1006.4±103.2)倍,随着培养时间的延长,细胞数量不再增加。CD34~+细胞低度表达端粒酶活性和hTERT基因,在细胞诱导分化过程中端粒酶活性及hTERT表达逐渐升高,细胞诱导14天后端粒酶活性及hTERT表达下降,28天端粒酶活性及hTERT基因检测不出。结论:利用CD34~+细胞在体外定向诱导分化出的大量细胞,不具有无限增殖的特性,可安全地应用于临床,且利用端粒酶的检测可为临床应用诱导分化细胞的时机提供依据。     

AZT对白血病细胞株KG-1a细胞增殖和端粒酶活性的影响

本研究探索端粒酶抑制剂3’-叠氮-2’,3’-脱氧胸腺核苷(AZT)对人急性髓系白血病细胞株KG-1a细胞增殖和端粒酶活性的影响。用MTT法检测不同浓度AZT分别作用24、48、72 h时KG-1a细胞增殖水平;流式细胞术检测AZT对KG-1a细胞周期及细胞凋亡的影响;TRAP-PCR-ELISA法检测细胞端粒酶活性;RT-PCR法检测细胞端粒逆转录酶(hTERT)mRNA的表达。结果表明,AZT能抑制KG-1a细胞增殖,抑制作用具有时间和浓度依赖性;随AZT浓度的增加,处于S期的细胞数目减少,G2/M期细胞数目增加,且出现凋亡峰;AZT作用后实验组细胞的端粒酶活性降低,hTERT-mRNA表达下降,下调程度与AZT浓度呈正相关。结论:AZT在体外能明显抑制KG-1a细胞增殖,并诱导其凋亡,其机制与降低端粒酶活性、下调hTERT mRNA表达有关。     

Characterization of a new monoclonal antibody 6G7 that recognizes a unique antigen on myeloid and lymphoid cells

We generated a monoclonal antibody (mAb) 6G7, which recognizes a 220-kD antigen on selected subpopulations of normal myeloid and lymphoid cells and their malignant counterparts. 6G7 reacts with 90-95% of peripheral blood B cells, 70-80% of CD8(+) cells, 30-35% of CD4(+) cells, 20-40% of monocytes, and 20-40% of CD34(+) cells from bone marrow. 6G7 reacts with leukemic blasts in acute myeloid leukemia (14/16), adult acute lymphoblastic leukemia (ALL) (5/5), pediatric ALL (5/9), chronic lymphocytic leukemia (8/8), follicular lymphoma (7/7), and Burkitt's lymphoma (1/1). Long-term bone marrow culture of 6G7(+/-) cells showed the majority of clonogenic hematopoietic cells were in mAb 6G7 subpopulation. An immunotoxin of 6G7 and ricin A chain was cytotoxic to 6G7(+) leukemia cell lines. mAb 6G7 has potential clinical applications for targeted immunotherapy of both leukemia and lymphoma.     

Diminished expression of CD19 in B-cell lymphomas

BACKGROUND: CD19 is expressed on most B-cell lymphomas; however, the frequency and types of B-cell lymphomas with low-level expression of CD19 are not well characterized. METHODS: We reviewed flow cytometric histograms specifically for decreased CD19 expression on 349 cases analyzed by the Flow Cytometry Laboratory at University Hospitals of Cleveland (Cleveland, Ohio). Results of flow cytometry were correlated with the morphologic diagnosis. RESULTS: Of the cases reviewed, 125 (36%) showed a visible decrease in CD19 expression compared with normal B lymphocytes. Decreased CD19 expression was noted in 79% of follicular lymphomas (27 of 34), 36% of small lymphocytic lymphomas/chronic lymphocytic leukemias (82 of 228), 31% of mantle cell lymphomas (4 of 13), 24% of diffuse large B-cell lymphomas (8 of 33), and 13% of marginal zone B-cell lymphomas/lymphoplasmacytoid lymphomas (4 of 30) and was not observed in any Burkitt lymphoma (0 of 5) or hairy cell leukemia (0 of 6). Decreased CD19 expression was significantly more frequent in follicular lymphomas than in other lymphoma subtypes (P < 0.001). No significant difference was observed in the frequency of decreased CD19 expression based on histologic grade of follicular lymphoma. CONCLUSIONS: Diminished expression of CD19 expression occurs frequently in B-cell lymphomas, in particular follicular lymphoma, and may be helpful in identifying B-cell lymphoma cells in complex cell mixtures such as bone marrow specimens.     

Phenotypic classification of malignant lymphoma

The phenotypic characters of the lymphoma cell are important in the diagnosis of this disease. We recently tested whether the flow cytometry with fresh biopsy sample might be useful in the diagnosis of the lymphoma. In our date, 1. a rise and fall of the surface immunoglobulin kappa/lambda ratio indicated the monoclonal proliferation of the B-cell, 2. the increased proportion of the CD5/CD23 double positive cells indicated B-cell chronic lymphocytic leukemia or small lymphocytic lymphoma, 3. the decreased proportion of the CD2 positive cells and the increased proportion of the CD19 positive cells indicated B-cell lymphoma. These findings suggest that the flow cytometry is of adjunctive importance in making a diagnosis of the lymphoma.     

Guess what: Chronic 13q14.3+/CD5-/CD23+ lymphocytic leukemia in blood and t(11;14)(q13;q32)+/CD5+/CD23- mantle cell lymphoma in lymph nodes!

We report a case of a patient with two B-cell lymphoproliferative disorders: CD5(-)/CD23(+) B-cell chronic lymphocytic leukemia and CD5(+)/CD23(-) mantle cell lymphoma. These disorders were diagnosed simultaneously based on flow cytometry, immunohistochemistry, fluorescence in situ hybridization, and polymerase chain reaction-based molecular studies. The B-cell lymphocytic leukemia clone predominated in the blood and bone marrow, whereas the mantle cell clone predominated in lymph nodes.     

Circulating levels of soluble CD23 reflect clinical and biological features of leukemic B-cell chronic lymphoproliferative disorders

Summary One hundred and twenty-four sera, from patients with various leukemic B-cell chronic lymphoproliferative diseases were investigated at diagnosis by ELISA for their soluble CD23 content. Immunophenotyping was carried out in all patients, and in a selected subset the mean number of membrane-bound CD23 molecules per cell was also investigated. Seventy-three patients had typical B chronic lymphocytic leukenia, 41 leukemic B-cell disorders with atypical morphological and/or immunophenotypic features, 5 had low-grade follicular cell lymphoma in the leukemic phase, and 5 had splenic lymphoma with villous lymphocytes Soluble CD23 levels were significantly higher than in normal sera (mean±SD: typical B chronic lymphocytic leukemia 3,650±4,654 U/ml, atypical B chronic lymphocytic leukemia 3,440±4,671 U/ml, follicular cell lymphoma 3,200±1,511 U/ml, splenic lymphoma with villous lymphocytes 8,236±7,294 U/ml, controls 137±128 U/ml;P<0.001). More advanced Rai's stages were related to higher soluble CD23 levels (P<0.01), both in typical and atypical B chronic lymphocytic leukemias, the highest levels and the best correlation with the absolute number of circulating CD19+ cells (r=0.50) being observed in the typical form. The number of membrane-bound CD23 molecules per cell was significantly higher in typical than in atypical B chronic lymphocytic leukemias (mean number 156,727±94,668 vs. 12,010±10,643,P<0.001). Our data suggest that soluble CD23 levels correlate with the clinical and biological features of leukemic B-cell lymphoproliferative disorders.     

Early neutrophil engraftment following autologous BMT provides a functional predictor of long-term hematopoietic reconstitution

BACKGROUND: Previous studies have demonstrated that the number of CD34+ progenitor cells in the stem cell graft is highly predictive of the rapidity of short-term hematopoietic engraftment. The aim of this study was to identify factors that predict long-term hematopoietic reconstitution (LHR) following autologous BMT. STUDY DESIGN AND METHODS: To identify predictors of LHR, peripheral blood counts and marrow biopsies were evaluated at 1 year after transplant in 81 patients with B-cell non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia who underwent autologous BMT. Results were correlated with CD34+ cell dose, granulocyte-monocyte colony-forming units (CFU-GM) infused, and time to neutrophil engraftment (TNE). RESULTS: Total MNC dose, CD34+ cell dose, and CFU-GM infused were significantly associated with TNE (p = 0.011, p < 0.0001, and p = 0.078, respectively). Patients with chronic lymphocytic leukemia were more likely to have received a low CD34+ cell dose than patients with B-cell non-Hodgkin's lymphoma (p = 0.01). Among the four principal predictors, only TNE showed consistent significant correlation with WBC, absolute neutrophil, and platelet count at 1 year after transplant. Logistic regression model showed that TNE was a more sensitive predictor of LHR than either CD34+ cell dose or CFU-GM infused. CONCLUSION: TNE is an in vivo functional measure of LHR and is a more sensitive predictor of LHR at 1 year after BMT than either CD34+ cell dose or CFU-GM infused.