骨髓成纤维细胞条件培养液对体外巨核系细胞生成的促进作用

目的：探讨骨髓成纤维细胞条件培养液对体外巨核系细胞生成的促进作用。方法：制备骨髓成纤维细胞条件培养液(bone marrow fibroblasts conditioned medium ,BMF-CM),分别观察BMF-CM或BMF-CM联合IL-11对体外培养条件下成熟巨核细胞生成的影响及BMF-CM对巨核系祖细胞生成的影响,并用RT-PCR的方法检测Meg-01人巨核细胞系细胞NF-E2 mRNA的表达。结果：BMF-CM促进成熟巨核细胞生成。BMF-CM联合IL-11促进成熟巨核细胞生成的效果更佳。BMF-CM能促进巨核系祖细胞的生成。将30％BMF-CM加入Meg-01人巨核细胞系培养体系培养4　h,与对照组相比,NF-E2表达增强。结论：骨髓成纤维细胞条件培养液对体外巨核系细胞生成有促进作用。     

人巨细胞病毒体外感染巨核系细胞加重凋亡

为了研究人巨细胞病毒 (HCMV)感染对巨核系细胞加快凋亡的作用机制及HCMV感染引起血小板减少症的机制 ,采用巨核细胞株CHRF 2 88 11和HCMVAD16 9株共同培养 ,用PCR检测HCMVIEA ,用形态学观察、DNALadder形成及AnnexinV/PI流式细胞仪检测分析细胞凋亡情况。结果显示 :HCMVAD16 9株的不同浓度病毒组(10 -3 ,10 -2 ,10 -1)均能显著抑制CHRF细胞的生长。在感染 7天后 ,3组细胞的活率水平分别是 77% ,73%和 6 8% ,而对照组为 98%。用流式细胞仪检测AnnexinV/PI,在感染 7天后 ,10 -3 ,10 -2 和 10 -1病毒组凋亡细胞的百分数是(2 1.3± 2 .4 9) % ,(2 5 .8± 3.6 5 ) %和 (31.4± 3.91) % ,对照组则为 (3.6 8± 1.4 7) %。凋亡率随培养液中病毒浓度的加大和感染后时间的延长而增高 ,二者呈依赖关系。形态学观察和DNALadder的形成进一步证实了凋亡细胞的存在 ,用PCR确定了在CHRF细胞内有HCMVIEA的表达。结论 :HCMV可直接感染巨核系细胞并加重它的凋亡。     

血小板生成素的研究进展

根据近年来的文献报道，概述了血小板生成素（ＴＰＯ）的研究进展。人ＴＰＯ基因长度约为６．２ｋｂ，含有６个外显子和个内含子。所有外显子－内含子的连接符合ＧＴ／ＡＧ规则。其ｃＤＮＡ克隆由１７７４个核苷酸附加一个多聚腺苷酸尾巴构成，它是一个１０５９个核苷酸的可译框架，是一种单拷贝基因。转录起始位点位于其ＤＮＡ－１９４９位的胞嘧啶外，在转录起始位占上游１．４ｋｂ的序列中没有通常哺乳动物启动子的基序和ＴＡＴＡ     

小儿急性淋巴细胞性白血病骨髓中巨核细胞变化

报告60例急性淋巴细胞性白血病患儿骨髓中巨核细胞的变化。有18.3%病例的巨核细胞数不减低。说明小儿急性淋巴细胞性白血病巨核细胞数不减低者并非罕见。在巨核细胞数量正常和增高者中,巨核细胞幼稚型比例增高,颗粒型明显增高,产血小板型明显低于正常。大多数病例骨髓片中血小板减少。表明巨核细胞存在成熟障碍。同时发现巨核细胞出现形态学方面改变。     

血小板生成素基因注射促血小板生成作用的实验研究

目的：研究血小板生成素（ＴＰＯ）基因注射法对健康小鼠血板的促生成作用。方法：将带有ｈＴＰＯ ｃＤ－ＮＡ的ｐｃＡＮＤ３质粒按６μｇ／只剂量注射至７５只昆明小鼠后肢内侧肌肉内，每天断尾采血计数白细胞和血小板，观察不同时间的骨髓和脾脏组织学变化。周期末注射小鼠做为对照组。结果：在基因注入后的３ｄ起即出现血小弧和巨核细胞增多。在所观察的２７ｄ内，血小板有一可持续１周的高水平期，平均约为同期对照的１８４％，     

Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano

Summary.  Background: Although mutations of GPIbα are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined. Objective: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIbα (Bolzano mutation). Methods: Human megakaryocytes were obtained by differentiation of patient cord blood-derived CD34⁺ cells and peripheral blood-derived CD45⁺ cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients' blood platelets. Moreover, α-tubulin distribution within proplatelets was severely deranged. Conclusions: Megakaryocytes from patients carrying a Bolzano allele of GPIbα display both quantitative and qualitative abnormalities of proplatelet formation in vitro . These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIbα in proplatelet formation.     

Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis

Idiopathic myelofibrosis, or agnogenic myeloid metaplasia, is a chronic myeloproliferative disorder characterized by clonal expansion and marrow fibrosis. Although marrow fibrosis appears to be a reactive process, it substantially contributes to impaired haemopoiesis. During the last few years the implication of megakaryocyte-derived growth factors in its pathogenesis has been documented.
We previously reported increased expression of TGF-β in patients with idiopathic myelofibrosis. In the present study we show that circulating megakaryocytic cells from such patients expressed high levels of basic fibroblast growth factor (bFGF). An increased expression of bFGF was also detected in patients' platelets. Under culture conditions, bFGF present in megakaryocytic cells was not exported into the medium, consistent with the fact that bFGF is devoid of a secretion peptide signal. Interestingly, this lack of bFGF secretion was observed in all patients but one, who was in an accelerated phase of the disease and presented an important percentage of circulating megakaryoblasts.     

Bone marrow macrophages and megakaryocytes express common acute lymphoblastic leukaemia antigen

Common acute lymphoblastic leukaemia antigen (CALLA) was demonstrated on a proportion of bone marrow macrophages and megakaryocytes. CALLA was detected by two monoclonal antibodies (J5 & BA3) in a three-layer immunoalkaline phosphatase system applied to routine air-dried bone marrow smears. The J5 staining was confirmed by an indirect immunofluorescent method and the CALLA was shown to be at the surface of the macrophages and megakaryocytes by an indirect immunogold technique. The findings are discussed in relation to the known tissue distribution of CALLA and to the clinical use of anti-CALLA antibodies for bone marrow purging.     

Accessibility of abciximab to megakaryocytes and endothelial cells in the bone marrow compartment: studies on a patient receiving antithrombotic therapy

Abciximab, chimaeric Fab fragments of the monoclonal antibody 7E3 (c7E3 Fab), has achieved widespread use as an anti-platelet agent for blocking GP IIb-IIIa (alphaIIbbeta3) function and preventing ischaemic complications after coronary artery angioplasty. However, its accessibility to the bone marrow compartment during therapy is unknown, as is its ability to bind alphavbeta3 in vivo. Using electron microscopy and immunogold labelling, we have looked for abciximab in the bone marrow of a patient who became thrombocytopenic during treatment. The presence of abciximab was assessed on ultrathin frozen sections of a marrow aspirate, the drug being revealed by a rabbit antibody to c7E3 Fab. Labelling was maximal on fragmenting megakaryocytes (MK) and proplatelets in the vascular sinus and in direct access to the blood compartment. Not only the plasma membrane but also the demarcation membrane system (DMS) and the membranes of alpha-granules were labelled. Abciximab was also revealed on the luminal surface of endothelial cells lining the marrow sinuses, thereby confirming for the first time its ability to bind to alphavbeta3 in vivo. The study revealed no signs that abciximab had accumulated in the marrow.