恶性髓系血液病患者血管内皮生长因子表达的检测及分析

目的探讨血管内皮生长因子(VEGF)在急性髓系白血病(AML)和骨髓增生异常综合征(MDS)患者中血管生成的作用。方法2002-06～2004-01南华大学第一附属医院用酶联免疫吸附双抗体夹心(ELISA)法检测初治20例AML及24例MDS患者骨髓单个核细胞培养上清VEGF的表达。结果AML患者骨髓细胞VEGF(412.30ng/L)均高于对照组(128.14ng/L),化疗后完全缓解组骨髓细胞VEGF明显下降(P<0.05),化疗后未缓解组的VEGF与治疗前比较无显著下降(P>0.05)。MDS高危型(RAEB或RAEBt)骨髓细胞VEGF与低危型(RA或RAS)及对照组相比明显升高(P<0.05)。其中RAEBt与AML患者相比差异无显著性(P>0.05)。结论AML及高危型MDS患者骨髓中存在血管新生,且VEGF在AML及MDS发病中起着重要作用,VEGF的表达与MDS病程进展有关。     

老年急性髓系白血病患者周围血清血管内皮生长因子水平的检测及意义

目的探讨血管内皮生长因子(VEGF)与老年急性髓系白血病(AML)发病及预后的关系。方法利用ELISA方法测定37例老年急性髓系白血病(AML)和12例健康对照者血清中VEGF水平。结果AML患者血清VEGF的含量(925.71±363.63)ng/L明显高于正常对照组(105.62±34.63)ng/L(P<0.001),VEGF水平高的AML患者与VEGF水平低的AML患者比较其CR率低、CR平均持续时间短(P<0.01)。结论VEGF与老年AML发病关系密切,血清VEGF水平的高低可预测老年AML治疗效果和预后。     

急性白血病患者血管内皮生长因子及其受体表达的动态观察

目的探讨急性白血病(AL)患者治疗前后骨髓中血管内皮生长因子(VEGF)及其受体的表达差异以及这种表达与血管生成的相关性.方法应用EnVision免疫组织化学二步法,检测122例次成人AL患者骨髓中造血细胞VEGF及其两种特异性受体fms-样酪氨酸激酶受体(Flt-1)、激酶插入嵌合受体(KDR)蛋白的表达情况.结果化疗后获得完全缓解(CR)的患者,其VEGF、KDR蛋白的表达在治疗前为6.0(3.3～12.0)和5.3(3.3～8.0),获CR后为5.3(3.3～9.0)和2.0(1.0～4.0)差异有显著性(P<0.05;P<0.01),而在化疗后未获得CR患者中的表达差异无显著性.在缓解后复发患者中的表达又升高到初发时的水平.各组初发患者Flt-1的表达水平与对照组之间差异无显著性,但CR期Flt-1的表达水平在CR组为3.3(1.7～5.3),复发组为3.3(2.0～5.3)与初发及对照组差异有显著性(P<0.01).微血管数处于高水平组的VEGF及KDR表达显著高于微血管处于低水平组者(P<0.01).骨髓原始细胞与急性髓系白血病(AML)初发患者VEGF和KDR的表达之间成正相关(r=0.429,0.359;P=0.005,0.02);与急性淋巴细胞白血病(ALL)初发患者VEGF的表达之间成正相关(r=0.522,P=0.03).结论 VEGF及其两种特异性细胞受体Flt-1, KDR在造血细胞及血管内皮细胞中表达.提示VEGF可能是白血病细胞的一种自分泌因子,同时作为一种旁分泌因子调控患者骨髓中的血管新生反应.VEGF及其细胞受体KDR可能构成抗血管新生和抗白血病治疗的新靶点.     

骨髓增生异常综合征患者细胞免疫功能及可溶性肿瘤坏死因子受体的研究

目的:研究骨髓增生异常综合征(MDS)外周血T细胞亚群分布及血清、骨髓可溶性肿瘤坏死因子受体1和2(sTNF-R1和sTNF-R2)的水平及其意义。方法:用流式细胞仪检测外周血T淋巴细胞CD3、CD4和CD8的表达,ELISA法检测血清和骨髓sTNF-R1和sTNF-R2的水平。结果:低危组(RA RAS)的CD3 细胞(70．04±4．25)％明显升高,MDS患者CD4 细胞减低,低危组为(30．77±6．58)％;高危组(RAEB RAEB-T)为(25．32±7．93)％,MDS患者CD4／CD8比例亦减低,低危组为0．89±0．24;高危组为0．91±0．52,而MDS患者CD8 细胞明显升高,低危组为(34．96±5．43)％;高危组为(29．67±7．38)％。MDS 2种sTNF-R水平均明显升高,低危组sTNF-R1血清为(1 586．13±573．24)ng／L,骨髓为(2 103．42±259．27)ng／L;sTNF-R2血清为(1 445．73±498．22)ng／L,骨髓为(1 961．50±614．53)ng／L;高危组sTNF-R1血清为(2 621．20±546．65)ng／L,骨髓为(3 146．03±412．14)ng／L;sTNF-R2血清为(1 972．34±558．31)ng／L,骨髓为(3 087．12±569．42)ng／L。血清sTNF-R2水平与CD8 细胞呈正相关,与CD4／CD8呈负相关。结论:细胞免疫异常及免疫分子表达异常增高,在MDS发病中起重要作用。     

多发性骨髓瘤患者骨髓微血管密度和血清β2-微球蛋白检测的临床意义

目的检测多发性骨髓瘤(MM)患者骨髓中血管新生的状态,探讨骨髓微血管密度(MVD)和β2-微球蛋白(β2-MG)与MM发展的关系。方法应用改良的塑料包埋骨髓活组织病理切片和免疫组织化学染色检测患者骨髓MVD;胶乳增强免疫透射比浊法测定MM患者血清β2-MG。结果初诊MM患者骨髓MVD明显高于正常对照组(P<0.01),与国际预后分期(ISS)及血清β2-MG相关(P<0.01),随临床分期的增加依次增高(P<0.01)。与治疗前相比,MM治疗有效组骨髓MVD水平明显下降(P<0.01),治疗无效组差异无统计学意义(P>0.05)。结论骨髓MVD及血清β2-MG检测可作为MM患者体内瘤负荷、预后及疗效判断的指标,骨髓血管新生在MM发生、发展过程中发挥重要作用,抗血管新生治疗可能成为治疗MM的新策略。     

急性白血病患者骨髓MVD与血清 VEGF水平的研究

目的：探讨急性白血病(AL)骨髓血管新生的状态及意义。方法：应用改良的GMA塑料包埋骨髓病理切片进行免疫组化染色检测AL患者治疗前后骨髓微血管密度(MVD)，应用ELISA方法检测AL患者治疗前后血清血管内皮生长因子(VEGF)的浓度。结果：AL患者治疗前骨髓MVD明显增加，化疗缓解后骨髓MVD降至正常水平，复发者骨髓MVD再次升高至化疗前水平；AL患者治疗前血清VEGF水平比正常对照明显升高，化疗缓解的患者VEGF水平仍然较高。结论：骨髓血管新生在AL发生过程中有着重要的作用，血清VEGF水平能够在一定程度上解释白血病骨髓血管新生现象。     

CHG方案治疗急性髓性白血病及高危骨髓增生异常综合征患者的疗效分析

目的:评价CHG方案治疗低增生、难治、复发急性髓性白血病(AML)及高危骨髓增生异常综合征(MDS)患者的疗效及不良反应.方法:对25例次AML和高危MDS患者实施CHG方案化疗.治疗前、治疗中及治疗后进行心电图、肝肾功能、外周血常规及骨髓细胞学检查,评估近期疗效及不良反应.结果:完全缓解率68%,部分缓解率8%;主要...     

骨髓粒细胞与红细胞表面CD55 CD59缺失对血液疾病的诊断意义研究

目的探讨骨髓粒细胞、红细胞表面糖基化磷脂酰肌醇(GPI)锚定蛋白CD55、CD59缺失(CD55-、CD59-,也称PNH细胞)在血液系统疾病中的意义。方法采用流式细胞仪检测中山大学附属第一医院血液科2008年9月至2010年11月诊治的正常人、阵发性睡眠性血红蛋白尿、再生障碍性贫血(AA)、骨髓增生异常综合征(MDS)、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)及营养不良性贫血患者外周血及骨髓中红细胞和粒细胞CD55、CD59缺失,并对结果进行分析。结果正常人骨髓粒细胞CD55-高于外周血(P<0.05);PNH患者骨髓红细胞CD55-、CD59-高于外周血(P<0.05);正常人、AA、MDS、AML、MM及营养不良性贫血各组间骨髓粒细胞CD55-表达无显著差异(P>0.05)。结论单一骨髓粒细胞CD55-表达升高特异性差。     

骨髓增生异常综合征患者骨髓血管新生及与临床特征的关系

目的探讨骨髓增生异常综合征(MDS)患者骨髓血管新生及与临床特征的相关性。方法收集2008年5月至2014年5月该院收治的78例MDS患者以及39例急性非淋巴细胞白血病(ANLL)患者,并于同期随机抽取80例健康体检者作为对照组,采用免疫组织化学法分别对三组的骨髓活检标本进行染色并计数微血管数,同时分析其与临床特征的相关性。结果 MDS组、ANLL组以及对照组的骨髓微血管计数经秩和检验,差异有统计学意义(P0.05),MDS组骨髓微血管计数高于对照组而低于ANLL组。MDS低危组骨髓微血管计数低于高危组(P0.05)。MDS患者骨髓微血管计数与性别、年龄、外周血中的白细胞、血小板以及血红蛋白含量无明显相关性(P0.05),与骨髓涂片中的原始细胞百分比有正相关性(r=0.386,P0.05)。结论 MDS患者骨髓中出现不同程度的血管新生,且骨髓原始细胞百分比越高,血管新生数量越多,对临床治疗MDS具有一定的指导意义。     

骨髓增生异常综合征CD34+细胞焦亡相关分子焦孔素E研究

目的 探讨骨髓增生异常综合征(MDS)患者骨髓CD34⁺细胞中焦孔素E(GSDME)与胱天蛋白酶-3(Caspase-3)表达水平。方法 选取2020年9月至2021年12月在天津医科大学总医院血液科收治的32例初治MDS患者与20例正常者对照,利用流式细胞术、qRT-PCR技术检测骨髓CD34⁺细胞中GSDME与Caspase-3表达水平。结果 流式细胞术结果显示：初治低危组骨髓CD34⁺细胞中GSDME与活化Caspase-3的表达水平[(64.90±15.74)%与(31.83±17.99)%]显著高于对照组[(41.39±14.07)%与(24.11±16.28)%,P<0.05]。初治高危组GSDME与活化Caspase-3的表达水平[(30.69±14.62)%与(15.42±13.34)%]显著低于对照组与初治低危组(P<0.05)。qRT-PCR结果显示：高危MDS患者骨髓CD34⁺细胞GSDME基因相对表达量低于对照组(0.20±0.19比1.00±0.53,P<...     

Shwachman-Diamond syndrome: an inherited model of aplastic anaemia with accelerated angiogenesis

Bone marrow angiogenesis is increased in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but has not been studied in inherited or acquired marrow failure syndromes. Shwachman-Diamond syndrome (SDS) carries a high risk of MDS/AML and is characterised by marrow stromal dysfunction. Compared with controls, SDS patients without MDS/AML had higher marrow microvessel density. Stromal VEGF gene expression, stromal vascular endothelial growth factor (VEGF) secretion and VEGF levels in serum and marrow mononuclear cells were normal. Future studies should investigate the mechanism for increased angiogenesis in SDS, and whether SDS marrow, with its increased angiogenesis, promotes progression of malignant clones.     

Altered expression of protooncogenes during clinical course in an AML case transformed from MDS

The changes of expression of oncogenes in the mononuclear cells of MDS case was studied during his clinical course, in series. His bone marrow was considered to maintain its function partly in initial stage, since both peripheral blood and bone marrow responded to clinical episodes. However, his hematopoietic function was gradually impaired with the disease evolution to AML. We examined the expression of four oncogenes in the mononuclear cells of his three clinical stages, early RAEB-t, RAEB-t and AML, to study the cause of transformation from MDS to AML. Early RAEB-t cells expressed all oncogenes studied other than c-myb, while only c-myc was weakly observed in RAEB-t. AML cells expressed c-myc, c-jun and c-myb, except for c-fms. The expression of c-fms and c-jun of early RAEB-t was considered to reflect the monocytosis induced by infections, and the expressions of c-myb and c-myc of AML cells were regarded as one of malignant signs of tumor transformation. These findings suggest that the evolutional transformation of MDS to AML was affected by the altered expression of oncogenes.     

Vascular Endothelial Growth Factor Levels in Childhood Acute Lymphoblastic and Myeloblastic Leukemia

Angiogenesis has been associated with the growth, dissemination and metastasis and has been shown to be a prognostic. Although there are some data suggesting that angiogenesis may have a role in the pathophysiology of leukemia, its role in patient prognosis is yet to be defined. We analyzed the expression level of vascular endothelial growth factor (VEGF), an angiogenesis promoter and its possible- prognostic value in bone marrow samples at the time of diagnosis and remission of acute childhood leukemia patients. Besides 46 patients diagnosed as ALL or AML, 16 children were also included as a control group in the study. Our data have demonstrated that VEGF levels of AML patients were found higher than the control group statistically (P = 0.022). However we could not find any significant difference between VEGF levels of diagnosis and remission in both AML and ALL groups by blastic VEGF expression (P > 0.05). In this study the higher levels of VEGF in AML patients is one of the main findings although we were not able to assess any role of VEGF in predicting prognosis in pediatric leukemia patients by evaluating blastic cell VEGF expression. These results have demonstrated that the relationship between angiogenesis or angiogenesis promoters and hematological malignancies is not clear and simple as different methods or different cells beside different angiogenesis promotors are involved to these studies. So that not only tumor cells and their cytokines but also surrounding cells and their cytokines must be taken into consideration with the standardized study methods in the further studies to obtain a promising treatment approach.     

Regulation of angiogenesis in the bone marrow of myelodysplastic syndromes transforming to overt leukaemia

To investigate the regulatory mechanisms of angiogenesis in the development of myelodysplastic syndromes (MDS) and its progression to overt leukaemia (OL), bone marrow samples from control, paired samples from MDS patients before and after transformation to OL (MDS --> OL) and de novo acute myeloid leukaemia (AML) were analysed. Immunohistochemical staining showed a significant increase of bone marrow microvascular density (MVD) in MDS and de novo AML compared with controls. Surprisingly, in MDS, MVD significantly decreased upon transformation to OL, which was also significantly lower than the MVD of de novo AML. This evidence was strengthened by the pattern of angiogenic mediator gene expression, confirming the importance of various angiogenic mediators including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFalpha), hepatocyte growth factor (HGF) and the angiopoietin family of mediators (Ang-1 and Ang-2) as well as the receptors for angiogenic mediators, such as VEGF receptor 2 (VEGFR2) and the tyrosine kinase receptor, TIE2. By contrast, the anti-angiogenic mediator, transforming growth factor-beta (TGFbeta) exhibited significantly higher expression in the bone marrow of MDS --> OL, indicating the importance of this cytokine as the suppressive factor of angiogenesis in MDS. These findings indicate that the bone marrow microenvironment in MDS --> OL and de novo AML differs remarkably, suggesting the different efficacy of anti-angiogenic therapy between de novo AML and leukaemia secondary to MDS.     

MDS患者白细胞介素2受体表达研究

本实验以２８例ＭＤＳ（ＲＡ１８例、ＲＡＥＢ和ＲＡＥＢ－Ｔ１０例）和１０例ＡＮＬＬ为研究对象，采用ＡＰＡＡＰ和ＥＬＩＳＡ法检测患者外周血单个核细胞（ＰＨＡ刺激前后）的ＭＩＬ－２Ｒ和培养血清中ＳＴＬ－ＩＲ，结果表明：经ＰＨＡ刺激培养４８ｈ后，ＭＤＳ和ＡＮＬＬ患者Ｔａｃ抗原阳性率明显低于正常人（Ｐ＜０．０１）ＲＡＥＢ和ＲＡＥＢ－ｔ组Ｔａｃ＋率比ＲＡ低，与ＡＮＬＬ差异无显著性（Ｐ＞０．０５）；血清中ＳＴＬ－２Ｒ在ＭＤＳ各亚型中均高于正常对照组（Ｐ＜０．０５），其中以ＲＡＥＢ及ＲＡＥＢ－ｔ为著。认为ＭＤＳ患者免疫反应及监视能减弱；ＳＴＲ－２Ｒ与ｍＩＬ－２Ｒ无相关；ＩＬ－２Ｒ表达异常可能与造血抑制有关。     

Intravenous administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary zone after stroke in rats

We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1x10(6)) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (P<0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (n=12) treated with hMSCs compared with numbers in rats (n=12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (P<0.05) raised endogenous rat VEGF levels in the IBZ from 10.5+/-1.7 ng/mL in the control group to 17.5+/-1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (P<0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (P<0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.     

Angiogenesis in acute myeloid leukemia and myelodysplastic syndrome

Increased angiogenesis is important in the pathophysiology of solid tumors. Recent studies show that angiogenesis and angiogenic factors play an important role in hematological malignancies. Both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are associated with a substantial increase in vascularity in the bone marrow as well as increased levels of various angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, angiopoietin-1, platelet-derived growth factor, hepatocyte growth factor, epidermal growth factor, tumor necrosis factor-alpha, and transforming growth factor-alpha and transforming growth factor-beta. Most of these angiogenic factors appear to be secreted by the neoplastic hematopoietic cells and appear to promote the growth and proliferation of the leukemic cells in an autocrine fashion. More importantly, angiogenic factors play a role in the clinical behavior and outcome of both AML and MDS. Despite significant overlap between MDS and AML in many aspects, higher levels of cellular VEGF and lower levels KDR are seen in MDS than in AML. Antiangiogenic therapy may play a role in AML and MDS and some differences in response may exist between MDS and AML.     

急性白血病患者骨髓血管新生的研究及临床意义

目的　研究急性白血病 (AL)患者骨髓血管新生、骨髓液血管内皮生长因子 (VEGF)水平并探讨其临床意义。方法　采用免疫组化方法 ,以兔抗人Ⅷ相关抗原多克隆抗体标记骨髓内皮细胞 ,光学显微镜下计数骨髓全切片微血管数 ;用ELISA方法检测骨髓液VEGF水平。结果　骨髓微血管数在初发未治组 (2 2 82 /× 4 0 0视野 )明显高于正常对照组 (7 17/× 4 0 0视野 )及骨髓缓解组(8 5 7/× 4 0 0视野 ) (P值均 <0 0 0 1) ,而骨髓缓解组仍高于正常对照组 (P <0 0 5 ) ,初发未治组与复发难治组 (2 1 83/× 4 0 0视野 )之间的差异无显著性 (P >0 0 5 )。初发未治组中 ,骨髓微血管数在 2 3例急性淋巴细胞白血病 (ALL)患者 (2 3 0 9/× 4 0 0视野 )与 4 3例急性非淋巴细胞白血病 (ANLL)患者(2 2 37/× 4 0 0视野 )、FAB亚型M1～ 3 (2 2 91/× 4 0 0视野 )与M4～ 5患者 (2 1 4 6 /× 4 0 0视野 )之间的差异均无显著性 (P值均 >0 0 5 ) ,而骨髓微血管数与骨髓原始细胞百分数呈正相关性 (r =0 311,P <0 0 1)。骨髓液VEGF水平 ,在初发未治组 (188 88ng/L)显著高于骨髓缓解组 (78 74ng/L)和正常对照组 (79 5 2ng/L) (P值均 <0 0 1) ,而后两者之间的差异无显著性 (P >0 0 5 ) ;在ANLL组(2 70 12ng/L)显著高于ALL组 (1     

Vascular endothelial cell growth factor is an autocrine promoter of abnormal localized immature myeloid precursors and leukemia progenitor formation in myelodysplastic syndromes

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-alpha and interleukin-1beta from MDS BM-mononuclear cells and BM-stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.