hPer3 基因启动子甲基化检测在慢性粒细胞白血病急变监测中的临床意义

目的: 探讨慢性粒细胞白血病(CML)急变中 hPer3 (human period 3)基因启动子甲基化检测的临床意义以及诱导 hPer3 表达对CML细胞株K562的作用。方法: 应用甲基化特异性PCR(MS-PCR)和实时荧光定量PCR检测29例CML患者骨髓 hPer3 基因启动子甲基化状态和mRNA表达,以40例缺铁性贫血(IDA)患者骨髓作为对照。将pcDNA3.1- hPer3 表达质粒经脂质体介导转染CML细胞株K562,MTT法检测生长抑制率,Annexin V/PI染色法检测细胞凋亡。结果: IDA组、CML慢性期、CML加速期和CML急变期中, hPer3 启动子甲基化阳性率(例)分别为0%(0/40)、17.65%(3/17)、66.67%(4/6)和83.33%(5/6),CML急变期和加速期甲基化阳性率均高于CML慢性期,差异显著(χ²=8.44,P＜0.01;χ²=5.03,P＜0.05)。对照组和CML慢性期、CML加速期和CML急变期中, hPer3 mRNA表达分别为75.03±18.16和5.13±2.29、0.40±0.18、0.17±0.05,两两之间差异显著(P＜0.01)。与空载体转染组相比, hPer3 转染后各时点的细胞抑制率和凋亡率均升高(P＜0.01)。结论: CML中 hPer3 启动子高甲基化导致了基因表达沉默,可能作为CML急变的监测指标。 hPer3 的过表达能抑制CML细胞株增殖,并促进其凋亡。     

慢性粒细胞白血病与Ag—NOR异型颗粒及骨髓染色体畸变相关研究

目的：研究银染核仁形成区异型颗粒和骨髓染色体畸变与慢性粒细胞白血病（CML）的相关性。方法：采用银染核仁形成区（Ag-NOR)染色技术和骨髓细胞短期培养制备染色体技术,分析60例不同病期的CML患者和20例正常对照组的Ag-NOR异型颗粒和染色体变异情况。结果：银染核仁形成区染色表明,病例组Ag-NOR染色计数颗粒总面积（GA）明显高于对照组（P＜0．05）;Ag-NOR染色计数颗粒总面积（GA）、Ag-NOR颗粒数（GN）,GA／细胞核面积（NA）的比率均随病情进展而增加。GN平均值急变期明显高于慢性期（P＜0．05）。骨髓染色体核型分析结果表明60例CML患者中58例有t(9:22)(q34;q11）易位,其中加速期、急变期患者还有其它变异。结论：CML与Ag-NOR异型颗粒及髓髓染色体变异具有相关性。     

人类端粒酶逆转录酶基因反义核酸促进顺铂诱导HL-60细胞凋亡

目的:利用人类端粒酶逆转录酶(hTERT)基因的反义寡核苷酸(ASODN)抑制HL-60细胞的端粒酶活性后,探讨顺铂对HL-60细胞凋亡的影响。方法:采用聚合酶链反应-酶联免疫测定(PCR-ELISA)方法检测人工合成的反义核酸处理HL-60细胞前后端粒酶活性的改变;免疫荧光标记通过流式细胞仪检测hTERT蛋白表达水平;用DNA电泳及流式细胞仪计数检测细胞凋亡。结果:hTERT基因的ASODN作用于HL-60细胞后,细胞的hTERT蛋白表达及端粒酶活性表现不断降低;抑制HL-60细胞端粒酶活性后,顺铂可诱导HL-60细胞产生明显的DNA断片;ASODN与顺铂联合作用于HL-60细胞后的凋亡细胞百分率(38.62±3.45)%分别同正义寡核苷酸(SODN)与顺铂联合作用组(11.02±2.07)%、单用顺铂作用组(9.23±1.70)%进行比较有显著差异(P＜0.01)。结论:hTERT基因的ASODN能促进顺铂诱导HL-60细胞凋亡。     

宫颈癌新鲜组织和石蜡包埋组织中端粒酶检测比较

目的为了了解宫颈癌新鲜组织和石蜡包埋宫颈癌组织中端粒酶活性检测的差异。方法TRAP-PCR用于30例宫颈新鲜组织和40例石蜡包埋的宫颈组织中端粒酶活性的检测。结果宫颈癌新鲜组织端粒酶活性检出率为73.33%(22/30),石蜡包埋的宫颈癌组织检出率为27.50%(11/40)(P<0.001)。两组平均端粒酶活性分别为1.362±0.768和0.624±0.386(P<0.001)。结论宫颈癌新鲜组织端粒酶活性检出率显著高于石蜡包埋的宫颈癌组织,平均端粒酶活性有极显著差异。     

慢性粒细胞白血病和原发性骨髓纤维化患者bcr/abl融合基因表达及临床意义

目的探讨bcr/abl融合基因的表达水平在慢性粒细胞白血病(CML)和原发性骨髓纤维化(MF)患者的诊断、疗效及预后观察中的意义.方法采用荧光定量逆转录多聚酶链反应(RT-PCR)技术,对18例初、复治CML、7例MF患者的骨髓或外周血单个核细胞进行了bcr/abl融合基因检测,并对其中2例行异基因骨髓移植(allo-BMT)患者进行了短期随防.结果 13例慢性期(CP)CML,12例不同程度表达bcr/abl融合基因,占92.3%,3例加速期(AP),急变期(BC)CML,2例表达bcr/abl融合基因,占66.7%,2例患者allo-BMT后一年内未检出 bcr/abl融合基因,三者之间有非常显著差异P＜0.01;8例初诊患者WBC数及bcr/abl融合基因表达水平明显高于常规治疗组;7例MF患者有1例表达bcr/abl基因.结论 bcr/abl基因是CML发病的分子基础,定量检测bcr/abl融合基因对于CML的诊断、临床分型、疗效观察及预后判断有重要意义;MF患者可能与CML关系密切,对MF患者应进行跟踪观察.     

慢性粒细胞白血病肿瘤干细胞的免疫调节功能

目的 研究慢性粒细胞白血病(CML)骨髓来源的肿瘤干细胞的免疫学特征,比较其与正常人来源的间充质干细胞(MSC)是否存在免疫功能的异常.方法 分离正常人和CML患者骨髓中的MSC,MLR法检测其对T细胞增殖的影响,流式细胞术检测其对T细胞周期、凋亡的作用情况.结果 CML和正常志愿者骨髓来源的MSC的细胞形态和表型没有差异,CML患者来源的MSC抑制T细胞增殖能力和抑制T细胞停留在G0/G1期的作用均减弱(CML MSC组74.5%±1.2%,BMSC组94.0%±1.9%,P＜0.05),CML患者抑制T细胞凋亡的作用增强(CMLMSC组8.36%±1.31%,BMSC组14.10%±0.65%,P＜0.05).结论 CML患者骨髓来源的MSC存在明显的免疫调节功能缺陷,如果使用CML患者自体的MSC移植治疗可能不是一种很好的选择,对于CML患者最好是选用异基因的MSC移植.     

PI-3K在低氧大鼠肺动脉平滑肌细胞增殖中的表达

目的:研究低氧肺动脉平滑肌细胞(PASMC)增殖时磷脂酰肌醇3 激酶(PI-3K)的表达。方法:实验分为无血清培养基(SFM)组和低氧48 h(H48 h)组, 应用免疫组织化学法和流式细胞仪检测低氧PASMC。结果:SFM组和H48 h组PASMC细胞浆内均有PI-3K p110阳性表达, H48 h组(0.1891±0.0301)的表达明显高于SFM组(0.1025±0.0164, P＜0.05);PCNA在SFM组的少数PASMC细胞核内有阳性表达, H48h组(24.58±4.07)%的增殖指数(PI)明显高于SFM组(8.76±1.21)%(P＜0.05);流式细胞仪检测发现PASMC的S+G₂／M期细胞所占细胞总数的百分比在H48 h(27.15±5.43)%显著高于SFM组(10.64±1.52)%(P＜0.05)。结论:低氧可显著上调PI3K的表达, 提示PI-3K可能在低氧PASMC增殖中起重要作用。     

慢性髓性白血病患者β-catenin的表达及其临床意义

目的:比较β-catenin在慢性髓性白血病(CML)各期中的表达情况,并分析与bcr/abl及伊马替尼细胞遗传学疗效的关系,为探讨CML疾病进展机制及寻找新的治疗靶点提供理论依据。方法:采用RT-PCR和Western blotting方法,检测99例CML患者骨髓单个核细胞中β-catenin mRNA和蛋白质水平的表达,分析与bcr/abl的相关性;94例CML患者服用伊马替尼1年后FISH检测bcr/abl融合基因,分析β-catenin与伊马替尼细胞遗传学疗效的关系。结果:CML急变期、加速期患者β-catenin的表达明显增高(P0.01),而慢性期与正常人无明显差别(P0.05);β-catenin与bcr/abl表达水平无明显相关性(r=0.314,P0.05);未达主要细胞遗传学缓解的CML患者β-catenin明显增高(P0.01)。结论:CML疾病进展阶段β-catenin的表达明显升高,β-catenin的表达与bcr/abl无明显相关,而与伊马替尼疗效有关,β-catenin可能参与CML疾病进展机制,可作为新的治疗靶点。     

7,12-二甲基苯丙蒽诱导Wistar大鼠乳腺癌发生过程中端粒酶活性变化

目的　动态观察在乳癌发生过程中端粒酶活性的变化 ,明确端粒酶在乳癌发生中的意义。　方法　采用 7,12 二甲基苯丙蒽 (DMBA)诱导雌性Wistar大鼠乳癌 ,TUNEL法检测凋亡细胞 ,RT PCR ELISA法检测端粒酶活性。　结果　在乳癌发生过程中端粒酶活性逐渐增强 ,乳腺增生组端粒酶活性高于正常乳腺组 (P <0 0 1) ,乳癌组端粒酶活性高于乳腺增生组 (P <0 0 1)。单发乳癌组与多发乳癌组间端粒酶活性无明显差异 (P >0 0 5 ) ,乳腺增生组织中凋亡细胞多于乳癌组织中凋亡细胞 (P <0 0 1)。　结论　在乳癌发生过程中 ,乳腺凋亡细胞逐渐减少 ,端粒酶活性逐渐增强     

PCNA与慢性粒细胞白血病急变关系的研究

目的：探讨增殖细胞核抗原（PCNA）与慢性粒细胞白血病（CML）急变之间的关系。方法：采用流式细胞仪技术检测CML患者在慢性期和急变期以及治疗后外周血中的PCNA表达水平。结果：CML慢性期与正常对照组的PCNA表达差异有统计学意义（P〈0．01）,在急变期PCNA表达水平显著高于慢性期（P〈0．01）,而在治疗达完全缓解后PCNA表达水平显著降低。结论：PCNA表达增高可能是引起CML急变的一个重要原因。     

Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis

We measured circulating endothelial precursor cells (EPCs), activated circulating endothelial cells (aCECs), and mature circulating endothelial cells (mCECs) using four-color multiparametric flow cytometry in the peripheral blood of 84 chronic myeloid leukemia (CML) patients and 65 healthy controls; and vascular endothelial growth factor (VEGF) by quantitative real-time PCR in 50 CML patients and 32 healthy controls. Because of an increase in mCECs, the median percentage of CECs in CML blast crisis (0.0146%) was significantly higher than in healthy subjects (0.0059%, P<0.01) and in the accelerated phase (0.0059%, P=0.01). There were no significant differences in the percentages of CECs in chronic- or active-phase patients and healthy subjects (P>0.05). In addition, VEGF gene expression was significantly higher in all phases of CML: 0.245 in blast crisis, 0.320 in the active phase, and 0.330 in chronic phase patients than it was in healthy subjects (0.145). In conclusion, CML in blast crisis had increased levels of CECs and VEGF gene expression, which may serve as markers of disease progression and may become targets for the management of CML.     

The immunohistochemical staining pattern of Gab2 correlates with distinct stages of chronic myeloid leukemia

Grb2-associated binder 2 protein (Gab2) is a member of scaffold proteins, playing crucial roles in (receptor-) tyrosine kinase and cytokine signaling. Chronic myeloid leukemia cells with t(9;22)(q34;q11) express the Bcr/Abl fusion protein, which interacts with Grb2 and Gab2 signaling, thereby triggering hematopoietic cell proliferation. The aim of this study was to examine in detail the total and subcellular Gab2 protein expression in myeloid cells in bone marrow biopsies of patients with chronic myeloid leukemia in different disease stages. The study included 50 fixed bone marrow biopsies of controls (unaffected hematopoiesis, n = 11) and Bcr/Abl-positive chronic myeloid leukemia cases (n = 39) of different stages (chronic phase, n = 13; accelerated phase, n = 4; blast crisis, n = 11; complete remission, n = 11). Immunohistochemistry and quantitative evaluation of Gab2 staining in 600 myeloid cells/bone marrow biopsy were performed before statistical analyses. Immunohistochemistry revealed Gab2 expression in hematopoietic cells. Gab2-positive myeloid cells occurred significantly more frequent in chronic myeloid leukemia cases than in controls (P < .001) and appeared to markedly increase from chronic phase to accelerated phase to blast crisis. Importantly, within the distinct stages of chronic myeloid leukemia, a significant switch of Gab2-positive myeloid cells with cytoplasmic or nuclear/perinuclear Gab2 staining occurred: Nuclear/perinuclear Gab2-positive myeloid cells significantly increased from chronic phase to accelerated phase (P = .001) and from chronic phase to blast crisis (P < .001). Still, an overlap and, hence, a wider range of Gab2 staining patterns were seen between and within chronic myeloid leukemia stages, most likely reflecting a high plasticity of Grb2-associated binder 2 functions in the progression of chronic myeloid leukemia. In summary, the present study, for the first time, analyzed Grb2-associated binder 2 protein expression in bone marrow biopsies of patients with chronic myeloid leukemia in detail, demonstrating a novel and distinct Grb2-associated binder 2 staining pattern in normal and chronic myeloid leukemia bone marrow biopsies as well as in distinct chronic myeloid leukemia stages. Grb2-associated binder 2 immunohistochemistry may provide a valuable supplementary tool to routine histopathology and standard immunohistochemistry for classification and staging of (borderline) chronic myeloid leukemia bone marrow biopsies and hence improved therapeutic disease management.     

A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression

Fluorescence in situ hybridization for the BCR/ABL rearrangement in 138 bone marrow specimens from 59 Philadelphia(+) (Ph(+)) chronic myelogenous leukemia (CML) patients, 35 Ph(+) acute lymphoblastic leukemia (ALL) patients, and 57 Ph(-) ALL patients was used. Sixteen (27.1%) of the 59 CML patients had deletions of the residual ABL gene on the derivative chromosome 9. During the study period, 32 of the 59 CML patients progressed to blast crisis or accelerated phase. Of these, nine patients had residual ABL gene deletions on the derivative chromosomes 9 and 23 patients had no deletions. The mean duration from first diagnosis to blast crisis or accelerated phase for the nine patients with ABL deletions was 32.8 months, and for the 23 patients without ABL deletions, it was 62.4 months (P = 0.017). The overall survival time for the 16 patients with deletions was 32.8 months, and for the 43 patients without deletions, it was 60.1 months (P = 0.164). ABL deletions were not detected among the 35 ALL patients (17 with major BCR/ABL, 18 with minor BCR/ABL), and it appears that this deletion occurs rarely or not at all in Ph(+) ALL patients, which is in contrast to the CML patients (27.1%). However, we detected two ALL cases with ABL deletion but without BCR/ABL rearrangement among 49 Ph(-) ALL and 66 Ph(-) AML patients. In conclusion, patients with ABL deletions progress to blast crisis or accelerated phase in a significantly shorter time than do those without such deletions. It is therefore suggested that the ABL deletion is an indicator of a poor prognosis in CML.     

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.     

Natural killer cells in chronic leukemia. Function and markers

Twenty-two patients with chronic lymphocytic leukemia (CLL) and 14 patients with chronic myelogenous leukemia (CML) were studied with respect to natural killer (NK) cell activity and related cell markers (Leu 7 and Leu 11b). Significantly reduced NK cell activity was detected in peripheral blood from the patients with CLL. Similarly, a reduced number of cells with the markers Leu 7 and Leu 11b (CD 16) were detected in the same patients. Removal of the leukemic cells by centrifugation of cells forming rosettes with mouse erythrocytes led to an augmented, but not fully normalized, NK activity. This indicates that the low NK activity in CLL partly may be due to overgrowth of leukemic cells. However, in spite of the lymphocytic infiltration, the NK cell activity in the bone marrow of CLL patients did not differ significantly from that of normal controls. The patients with CML in the chronic phase as well as patients in the accelerated or blast phase also had a reduced NK activity. The finding that patients in the chronic phase had a reduced NK activity and normal numbers of Leu 11b (CD 16) positive cells, together with no detectable blasts in the peripheral blood, indicates that patients with CML may have an inherent NK cell defect. The highly reduced activity found in patients with the accelerated/blast form may in addition partly be due to overgrowth of leukemic cells. This low NK activity may be of importance in the development of chronic leukemia.     

Growth kinetics and blast-colony forming cell binding capacity of stromal cells in various haematological malignancies.

Growth kinetics of bone marrow stromal layers from normal, AML, ALL and CML patients was studied. Significantly reduced time for confluency was observed in AML patients in complete remission, in CML patients in chronic phase, or CML patients after allogenic bone marrow transplantation. The functional capacity of these stromal layers did not differ: they all bound similar amounts of blast colony forming cells (BL-CFC) from normal bone marrow. The stromal layers from bone marrow transplanted patients varied in their BL-CFC binding capacity: two CML patients (10.5 and 49 months after transplantation) showed normal values, while two ALL patients (1.5 and 3 months, respectively, after transplantation) as well as one patient transplanted for CML (19.5 months after transplantation) showed significantly reduced BL-CFC binding capacity.     

Stem cell regulation and the development of blast crisis in chronic myeloid leukemia: Implications for the outcome of Imatinib treatment and discontinuation

Chronic myeloid leukemia (CML) is a cancer of the hematopoietic system that is initiated by a single genetic alteration (the BCR-ABL fusion gene or Philadelphia chromosome) and progresses in several phases: during the chronic phase the number of cells grows slowly and the fraction of immature cells is low. During the accelerated phase and blast crisis, the population of CML cells and the fraction of immature cells rises sharply. The mechanisms that drive the transition from the chronic phase to blast crisis are not understood, and the requirement of genetic instability and further mutations has been suggested. Using mathematical models, I describe a theory that can account for the transition from the chronic phase to blast crisis without the need to invoke further mutations. The transition to blast crisis can be explained solely by feedback mechanisms that regulate the patterns of stem cell division, in particular the occurrence of symmetric versus asymmetric cell division. The model also has implications for the outcome of Imatinib treatment. According to the model, treatment can lead to the low level persistence of CML stem cells without assuming that these cells are less susceptible to drug-mediated activity, and this might explain why disease tends to relapse after treatment discontinuation even in the absence of acquired drug resistance. Further, the model defines conditions when Imatinib treatment might lead to the eradication of CML, which is relevant in the context of recent data that show absence of relapse as long as two years after treatment cessation.