重组红细胞生成素对小鼠巨核细胞后期成熟及血小板生成的影响

本文应用血小板生成液体培养体系,检测了重组人红细胞生成素(r-EPO)对巨核细胞成熟及血小板生成的影响。r-EPO 能在1U 至6~U/ml 浓度范围内增加体系血小板数,r-EPO剂量与血小板数之间呈线性关系。r-EPO 还能促进巨核细胞 DNA 合成,并使 Ⅱ、Ⅳ 期巨核细胞比例增加,Ⅰ、Ⅱ 期巨核细胞比例减少。结果表明:r-EPO 可以促进巨核细胞成熟,并作为一种主要刺激因子,以增加血小板数的方式促进血小板生成。     

肺的血小板生成功能

人们通常认为,血小板是从骨髓成熟的巨核细胞胞质裂解脱落下来的具有生物活性的小块胞质。虽然研究者早已发现在肺组织中存在大量巨核细胞,但肺是否具有生成血小板的功能一直存在争议。本文主要就肺的血小板生成功能假说的提出、早期研究过程、最新研究证据及可能的意义进行简要综述。     

血小板生成研究进展

血小板生成,即血小板颗粒由巨核细胞释放,进而在外周血逐步成熟的过程.随着解析血小板功能多样性以及高效再生策略的需求,对于血小板生成过程的全面认知显得尤为重要.然而,以往研究大多聚焦在造血干祖细胞和巨核细胞分化阶段,对于后期血小板生成过程的了解相对较少.该文对血小板生成过程中血小板形态、分子特征、功能的动态变化以及调控机...     

体外液体培养体系中生成的血小板性能观察

应用液体培养法培养小鼠骨髓细胞获得了比较稳定且有一定数量血小板生成的培养体系。培养3、5、7、9d时体系中的血小板数均高于接种时。在培养7d时可见巨核细胞生成血小板的现象,~(35)S掺入也证实体外有血小板生成。这些体外生成的血小板形态功能基本正常,其直径为1—5μm,新生成的血小板体积较大。无论体积大小,其活动性均稍强于正常。这些体外生成的血小板具有正常粘附功能,2×10~(-4)mol/L ADP可诱导出单波聚集。体系中血小板及巨核细胞生成量稳定且与接种细胞数呈正相关,提示可将其应用于巨核系生成调控的研究。进一步增加并稳定血小板生成量可使此体系更有效地应用于血小板形态、功能及生成调控的研究。     

巨核细胞生成与调控的研究进展

巨核细胞由骨髓造血干细胞分化为巨核系祖细胞,再经过渡细胞的增殖分化,成熟后生成血小板。这一过程受到巨核细胞集落刺激因子和血小板生成素的特异性刺激,白细胞介素３,粒单系集落刺激因子,白细胞介素６和红细胞生成素等细胞因子的非特异性刺激,及血小板蛋白因子、β转化生长因子和干扰素的抑制,以及辅助骨髓细胞的调控。     

巨核细胞系统生成的调控

巨核系是髓系造血的一部分,来自造血干细胞的巨核祖细胞,经增殖分化成为成熟的巨核细胞,最后生成血小板。巨核集落刺激因子主要作用于巨核祖细胞,使其增殖分化,并在体外增加巨核集落生成率。血小板生成索是肾脏等器官产生的糖蛋白,其血中含量受血小板数反馈调节,它的主要作用是促进巨核细胞DNA 合成、胞浆成熟和血小板生成。     

microRNA调控血小板生成及功能

microRNA（miRNA）是一类长约22nt（19～23nt）在进化上非常保守的非编码RNA,其能够通过降解mRNA或抑制mRNA翻译来调控蛋白表达。研究证实miRNAs在包括胚胎干细胞分化、单核细胞和巨核细胞生成等血细胞形成过程中扮演重要的角色。而由巨核细胞生成的血小板内同样存在miRNAs,并且被证实参与到血小板活化和血小板生理病理状态改变等过程中。对miRNA调控血小板生成及功能的深入研究将有利于早期诊断治疗血液系统相关疾病。     

血小板缺陷型动物血浆的制备

血小板减少症至今尚无特效约。肿瘤患者在接受放疗、化疗后往往由于血小板过低而中断治疗;骨髓患者也常常出现同样问题。巨核细胞的血小板生成受多方面因素的控制。其中血小板生成素(Thrombopoietm,TPO)是调节巨核细胞成熟,促进血小板生成的重要因子,是治疗血小板减少症的一种很好的潜在药。目前。国外重组TPO(rTPO)已进入临床试验阶段,国内未见报道。为配合rTPO的研究,特制备血小板缺陷型血浆。血小板最低值时。血浆TPO活性最高。文献已报道了用抗血小板抗体或用~(137)Csγ射线照射引起动物血小板减少的方法。我们采用~(60)Coγ射线照射引起动物血小板缺陷以刺激TPO的产生,进而制备有促进血小板增生活性的血浆即TPO。     

生物节律影响巨核细胞发育和血小板产生的研究进展

自然界中生物体的生命活动、生活习性都存在着一定的周期性变化。生物昼夜节律的产生是以内源性的生物钟系统为基础的。生物钟不仅易受到外界环境的影响,而且可以通过调控一系列特定的下游基因的表达,影响生物体的生理生化过程。巨核细胞是生成血小板的前体细胞,经过分化、增殖、成熟和裂解,最终生成血小板。血小板是一种没有细胞核的特殊细胞,在生理性止血和器官修复上发挥着重要作用,同时参与血栓等多种疾病的发生。近几年借助现代分子生物学和细胞生物学手段。证实了哺乳动物的巨核细胞和血小板的生成呈现明显的周期性的变化,利用生物钟基因缺失模型进一步发现了生物钟基因对巨核细胞和血小板的影响。本文概述了生物节律对巨核细胞和血小板的影响,为进一步研究巨核细胞的发育和血小板生成机制提供了参考。     

生物节律影响巨核细胞发育和血小板产生的研究进展

自然界中生物体的生命活动、生活习性都存在着一定的周期性变化。生物昼夜节律的产生是以内源性的生物钟系统为基础的。生物钟不仅易受到外界环境的影响,而且可以通过调控一系列特定的下游基因的表达,影响生物体的生理生化过程。巨核细胞是生成血小板的前体细胞,经过分化、增殖、成熟和裂解,最终生成血小板。血小板是一种没有细胞核的特殊细胞,在生理性止血和器官修复上发挥着重要作用,同时参与血栓等多种疾病的发生。近几年借助现代分子生物学和细胞生物学手段,证实了哺乳动物的巨核细胞和血小板的生成呈现明显的周期性的变化,利用生物钟基因缺失模型进一步发现了生物钟基因对巨核细胞和血小板的影响。本文概述了生物节律对巨核细胞和血小板的影响,为进一步研究巨核细胞的发育和血小板生成机制提供了参考。     

In vitro stimulation of megakaryocyte maturation by megakaryocyte stimulatory factor

Counterflow centrifugal elutriation and Percoll density gradient centrifugation were employed to prepare cell populations from rat bone marrow that were selectively enriched in the cytoplasmically immature megakaryocytes and depleted of the most mature megakaryocytes. The incorporation of [14C]leucine into the platelet-specific alpha-granule protein, platelet factor 4, as well as the incorporation of [35S]sulfate into platelet proteoglycans synthesized by the maturing megakaryocytes were monitored as markers of cytoplasmic maturation. Rat platelet factor 4 was specifically isolated and characterized by its high affinity for heparin-Sepharose and its amino-terminal sequence homology to human and rabbit platelet factor 4. The [35S]sulfate-labeled proteoglycans were primarily composed of chondroitin 4-sulfate glycosaminoglycans and were identified as platelet granule components by their ability to be secreted by megakaryocytes in response to thrombin or A23187. The production of both components was increased as much as 3-fold in a dose-dependent manner by the addition of picomolar concentrations of purified megakaryocyte stimulatory factor, without a concomitant increase in general protein synthesis. The above results suggest that the megakaryocyte stimulatory factor may regulate the synthesis of platelet granule components by megakaryocytes and hence control the rate and/or extent of cytoplasmic maturation during megakaryocyte development.     

Aspirin inhibits rat megakaryocyte thromboxane synthesis

This study examines the question of whether the aspirin-induced delay in the recovery of platelet cyclooxygenase pathway activity, as measured by RIA of thromboxane B₂, results from a direct effect on megakaryocyte cyclooxygenase. From our measurement of recovery of TXB₂ and information on megakaryocyte transit time in rats, we propose that thromboxane synthesis may represent a relatively late step in the differentiation of megakaryocytes. Megakaryocyte thromboxane production was depressed by 70% and that of platelets by 85% at two hr after 20 mg/kg oral aspirin dissolved in DMSO. Full megakaryocyte thromboxane recovery occurred by 72 hr and preceded complete platelet thromboxane recovery by 24 hr. Whereas megakaryocyte thromboxane synthesis showed substantial recovery by 36 hr after aspirin, platelet recovery did not begin for 24 hr and achieved a maximal recovery rate over the following 12 hr. This finding is consistent with predictions based upon human data for both megakaryocyte labeling studies and post-aspirin platelet recovery. We conclude from our data and from estimates of megakaryocyte maturation times in marrow, that thromboxane synthesis develops in rat megakaryocytes after approximately 48 hr of cytoplasmic differentiation toward platelet shedding. This metabolic capacity therefore serves as a marker of megakaryocyte differentiation.     

Stimulation of megakaryocytic spleen colonies in mice by thrombopoietin.

Kidney cell culture medium that was shown to stimulate thrombocytopoiesis in TSF assay mice was injected into lethally-irradiated, bone marrow transplanted mice. Results showed that the injection of TSF-rich material caused an increase in the number of megakaryocytic colonies when compared to control mice. The numbers of surface colonies and colonies/spleen section were not altered by TSF treatment. Erythroid, granulocytic, and undifferentiated colonies were not elevated by TSF injection. These data support the hypothesis that kidney cells in culture produce a humoral factor that controls the regulation of platelet and megakaryocyte production.     

Maturation and regulation of megakaryocytopoiesis.

The in vitro cloning technique for detecting megakaryocyte precursor cells was employed to compare stimuli known to influence megakaryocytopoiesis. Preparations of thrombopoietic stimulating factor (TSF) did not directly stimulate the growth of megakaryocyte colonies (CFU-m) but increased the frequency of CFU-m when TSF was added to the cultures with a constant amount of megakaryocyte colony stimulating factor. Platelets or platelet homogenates did not influence the frequency of CFU-m or the size of individual colonies. Analysis of cell surface properties of megakaryocytes obtained either by isolation from bone marrow or from in vitro colonies revealed species differences. The possibility that megakaryocytopoiesis and platelet release are regulated both within the marrow as well as by humoral factors is discussed.     

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.     

Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Delta60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.     

Human megakaryocytopoiesis--normal and abnormal.

Regulation of megakaryocyte and platelet production remains poorly understood. In culture system two separate activities are needed for maximum production of megakaryocyte progenitors: promotor of clonal expansion and promoter of maturation, other growth factors and cells also contribute to regulation of megakaryocytopoiesis. Increased proliferation of megakaryocytes is observed in myeloproliferative disorders and idiopathic thrombocytopenic purpura, and decreased proliferation is found in aplastic anaemia and hypomegakaryocytic thrombocytopenia. Dysmegakaryocytopoiesis is present in myelodysplastic syndromes and acute leukaemia, and a proliferation of immature megakaryocytes in acute megakaryoblastic leukaemia. Increased understanding of human megakaryocytopoiesis is beginning to help in rational clinical management.     

Mouse models of diseases of megakaryocyte and platelet homeostasis

Platelets are the small anuclear blood cells that are the product of megakaryocytopoiesis, the process of hematopoietic stem cell commitment to megakaryocyte production and the differentiation and maturation of these cells for platelet release. Deregulation or disruption of megakaryocytopoiesis can result in platelet deficiencies, the thrombocytopenias, with attendant risk of hemorrhage or thrombocytosis, a pathological excess of platelet numbers. Mouse models, particularly those engineered to carry genetic alterations modeling mutations associated with human disease, have provided important insights into megakaryocytopoiesis and deregulation of this process in disease. This review focuses on mouse models of diseases of altered megakaryocyte and platelet number, illustrating the profound contribution of these models in validating suspected roles of disease-associated genetic alterations, promoting discovery of new links between genetic mutations and specific diseases, and providing unique tools for better understanding of disease pathophysiology and progression, as well as resources to define drug action or develop new therapeutic strategies.