rhTPO 对60Co 照射小鼠造血系统影响的研究

目的为了检测ＴＰＯ的体内活性及其对血小板减少的治疗作用，寻找用以治疗辐射及化学药物等所致骨髓损伤造成的血小板减少与缺乏，缓解临床上的出血症状，降低死亡率的一种新途径。方法本实验应用基因工程的方法重组了人的ＴＰＯ，取昆明种正常雄性，经一次５．０Ｇｙ６０Ｃｏγ射线全身照射小白鼠为动物模型，通过对外周血血象的观察，及小鼠骨髓造血祖细胞培养的检测，研究了ｒｈＴＰＯ在体内对６０Ｃｏ全身照射处理小鼠的造血系统的影响。结果实验表明，ｒｈＴＰＯ能够减轻照射对小鼠外周血血小板及骨髓造血祖细胞中巨核系祖细胞的影响，并有促进其早期恢复的作用，且这种作用是量效相关的。结论提示，ＴＰＯ是巨核细胞系统较特异的正调控因子，具有促进巨核细胞增殖发育和刺激血小板生成的作用，是临床治疗骨髓组织损伤引起的血小板减少和缺乏及贫血的有希望的生物治疗药物。     

Significance of polyploidy in megakaryocytes and other cells in health and tumor disease

Summary Polyploidy — the doubling of chromosome sets of cells caused by a stop of mitosis at different levels of the mitotic cycle — is a phenomenon widely observed in plants, protozoa, metazoa, and animals. In man obligate polyploid tissues are found in liver parenchyma, heart muscle cells, and bone marrow megakaryocytes. Polyploidy occurs mostly in stable and highly differentiated cells and tissues. Besides age, stimulation of proliferation and increased metabolic function lead to polyploidization in these organs. Aneuploidy, however, is exclusively found in tumor cells.Megakaryocyte differentiation and polyploidy are controlled by thrombopoietin-like activities, of which the loci of production are still unknown. Megakaryocytes are unique among polyploid mammal cells. On the precursor level they maintain their proliferative activity independently of the mammal's age. Once having entered the incomplete mitotic cycle they stop cytokinesis and develop into highly polyploid cells. Polyploidization of megakaryocytes is the basic requirement for establishing highly effective hemostasis in mammals, which exhibit blood circulation based on high blood pressures.Every polyploidization results in increased production of membrane materials with which the platelet becomes endowed. By shedding cytoplasmic fragments approximately 3000 platelets are set free from a 32c megakaryocyte, compared with only 16 nucleated thrombocytes by mitotic division. There is further evidence that the heterogeneity of platelets mostly depends on the different polyploidy classes of the megakaryocytes from which they are derived. Changes in the polyploidy pattern of megakaryocytes could therefore have consequences for hemostatic disorders in several human diseases, particularly in malignancy.Abbreviations n haploid chromosome set (number of chromosomes actually counted) - c haploid DNA content (measured by cytophotometry) - M mitosis - C-Mitosis colchicine mitosis - G₀, G₁, G₂, S phases of the mitotic cycle - Meg CSF megakaryocyte colony stimulating factor(s) - TSF thrombocytopoiesis stimulating factor(s) - CFU-Meg megakaryocyte colony forming unit This work was supported by Deutsche Forschungsgemeinschaft, grant Wi 806/1-1     

Thrombopoietin, interleukin-6, and P-selectin at diagnosis and during post-steroid recovery period of patients with autoimmune thrombocytopenic purpura

 Plasma concentrations of the most potent megakaryocytopoietic cytokines, thrombopoietin (TPO) and interleukin-6 (IL-6), and the platelet activation marker P-selectin were evaluated in 24 patients with autoimmune thrombocytopenic purpura (ATP) who responded to conventional steroid treatment, at diagnosis and after steroid-induced recovery. Baseline TPO concentration (median [interquartile range]= 0 [17.52] pg/ml) was significantly decreased and IL-6 (38 [19.75] pg/ml) and P-selectin (485 [393.75] ng/ml) were significantly elevated compared with healthy subjects (100 [68] pg/ml, 8 [7] pg/ml and 166 [69] ng/ml, respectively). Following steroid treatment, all values approached normal, i.e., TPO (20 [18.75] pg/ml) was increased and IL-6 (19.5 [13] pg/ml) and P-selectin (248 [172.5] ng/ml) were decreased, significantly. The decrease of TPO in ATP is suggested to occur due to increased megakaryocyte mass and, consequently, TPO clearance. The non-lineage-specific cytokine IL-6 may be elevated to compensate for megakaryocytopoiesis/thrombopoiesis. The elevation of P-selectin may reflect compensatory platelet hyperactivation; however, this molecule also might be a marker of platelet destruction. Received: January 23 / 1998 · Accepted: May 12, 1998     

重组人血小板第4因子生物学活性的分析

目的：分析重组人血小板第４因子（ｒｈＰＦ４）的生物学活性。方法：采用小鼠骨髓巨核细胞以及人巨核细胞白血病细胞株，从体内和体外多方面比较天然ＰＦ４和ｒｈＰＦ４的生物学活性。结果：此ｒｈＰＦ４具有与天然ＰＦ４相同的抑制小鼠骨髓巨核细胞祖细胞生长、减少集落形成的能力，这种抑制作用能被肝素中和；ｒｈＰＦ４对人巨核细胞白血病细胞株Ｍｅｇ－０１生长也有抑制作用。结论：ｒｈＰＦ４可取代天然ＰＦ４应用于临床及科研。     

Hematopoietic stromal cells and megakaryocyte development

Abstract

The hematopoietic microenvironment, and in particular the hematopoietic stromal cell element, are intimately involved in megakaryocyte development. The process of megakaryocytopoiesis occurs within a complex bone marrow microenvironment where adhesive interactions, chemokines, as well as cytokines play a pivotal role. Here we review the effect of stromal cells and cytokines on megakaryocytopoiesis with the aim of exploring new therapeutic strategies for platelet recovery after hematopoietic stem cell transplantation (HSCT).     

Spontaneous proliferative megakaryocytopoiesis and platelet hyperreactivity in essential thrombocythemia: is thrombopoietin the link?

 Essential thrombocythemia (ET) is one of the less rare variants of the chronic myeloproliferative disorders (MPD). The present review questions the possible link between spontaneous megakaryocytopoiesis, platelet hyperreactivity, and the occurrence of platelet-mediated vascular manifestions in acquired and hereditary ET. In acquired ET, the role of thrombopoietin (TPO) is crucial to the observed hypermegakaryocytopoiesis, which is characterized by an increased proliferation of megakaryocyte (MK) progenitors, even in conditions of culture without addition of any known megakaryocyte colony-stimulating factors. An increased reactivity of megakaryocyte progenitors to TPO remains to be precisely delineated. A defective clearance of TPO by megakaryocytes and platelets because of a reduced number of TPO receptors is possible. TPO is able to enhance platelet aggregation induced by ADP, thrombin, and collagen. A point mutation in the TPO gene as the cause of increased TPO production in hereditary ET can readily explain both spontaneous megakaryocytopoiesis and platelet-mediated microvascular manifestations simulating the phenotype of acquired ET. Nevertheless, to date, no mutation of the TPO structural gene, as shown in two families with hereditary ET, and no mutations in the TPO receptor have been found in patients with acquired ET. There is good evidence that the microvascular circulation disturbances in ET are caused by intravascular activation and aggregation of hypersensitive platelets, with sludging or occlusion of the endarterial microvasculature. In this process, the generation of platelet-derived products, endothelial cell damage, fibromuscular intimal proliferation, and platelet thrombi are essential and can be inhibited by a platelet-specific regimen of aspirin, thus providing a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythemia. In contrast, the generation of thrombin appears not to be essential for the formation of platelet thrombi, thereby explaining the inefficacy of coumadin derivatives and heparin in the treatment and prevention of microvascular circulation disturbances in hereditary and acquired ET. Received: January 7, 1999 / Accepted: July 6, 1999     

Refractory thrombocytopenia with chromosome 11q23 abnormality

 Although cytopenia is a common manifestation of myelodysplastic syndrome (MDS), isolated thrombocytopenia is rare. The term "refractory thrombocytopenia" (RTC) has been proposed as a counterpart of refractory anemia. We describe here a case of RTC associated with chromosome abnormality on 11q23. A 59-year-old man was admitted because of severe thrombocytopenia. A bone marrow examination revealed an increased number of micromegakaryocytes and the absence of normal-sized megakaryocytes without obvious dysplasia in either the myeloid or the erythroid lineage. A remarkable increase of GPIIb/IIIa (CD41a)-positive precursor cells in the bone marrow was observed. Cytogenetic examination detected the chromosome abnormality, an addition on chromosome 11q23. Translocation of the HRX gene was not detectable by Southern blot analysis. The diagnostic problems of isolated thrombocytopenia and possible participation of gene(s) on chromosome 11q23 in megakaryocytopoiesis as well as early hematopoiesis are discussed. Received: 26 February 1996 / Accepted: 1 May 1996     

Anti-thoracic duct lymphocyte globulin stimulates human megakaryocytopoiesis in vitro

Summary Anti-thoracic duct lymphocyte globulin (ALG) therapy is effective in patients with aplastic anemia. We examined the effect of ALG on human megakaryocyte progenitor cells (colony-forming unit-megakaryocyte, CFU-Meg) in vitro. Normal human bone marrow mononuclear cells (MNC) were cultured in plasma clots with varying concentrations of ALG or non-immunized horse IgG. After 12 days of culture, significant megakaryocyte colony formation was observed in cultures containing ALG but not in cultures containing non-immunized horse IgG. The peak stimulatory effect seemed to occur with 10–25µg/ml of ALG. When marrow MNC, depleted of adherent and T cells, were cultured in plasma clots with ALG, its stimulatory effect on megakaryocytopoiesis decreased markedly. Finally, it was demonstrated that ALG stimulated marrow MNC to produce a factor stimulatory for CFU-Meg. The in vitro megakaryocytopoietic stimulatory effect of ALG may be related to its clinical efficacy in some patients with aplastic anemia.     

Improvement of megakaryocytic progenitor culture for toxicological investigations

The aim of this work was to obtain an in vitro test for the evaluation of xenobiotic toxicity on the proliferation and on the differentiation of megakaryocyte progenitors. The rapid rate of blood cell renewal makes the hematopoietic system a susceptible target for xenobiotic toxicity. Hematotoxic molecules can affect one or more hematopoietic lineages leading to blood disorders. Megakaryocytopoiesis in vitro models applied to toxicological investigations needs to be accurate, precise, reproducible, sensitive and specific. Human hematopoietic progenitors from umbilical cord blood were seeded in a collagen medium. Three solvents have been selected (ethanol, methanol, acetone), and one (dimethyl sulfoxide; DMSO) has been eliminated due to its cytotoxicity at tested concentrations. Cryopreservation did not affect the sensitivity of CFU–MK to xenobiotics. An overnight incubation of cell suspensions as cell suspension enrichment before plating gave better cloning efficiency than CD34⁺ cells negative selection. Comparison between different parameters allowed us to propose a protocol suitable for an in vitro megakaryocytopoiesis model in toxicological investigations. The effects of three toxins were studied on CFU–MK development in order to verify the efficiency of this clonogenic assays for toxicity testing. The CFU–MK culture conditions defined revealed their usefulness for investigating drug cytoxicity towards megakaryocytic progenitors and disturbance of their proliferation.     

Establishment of Megakaryoblastic Cell Lines by Coinfection of Abelson Murine Leukemia Virus and Recombinant SV40-retrovirus

Murine embryonic cells including yolk sac prepared from 8-day embryos were co-infected with Abelson murine leukemia virus (A-MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M-SV40). By coinfection with A-MuLV and M-SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A-MuLV or M-SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single-cell transfer, 6 acetyl-cholinesterase (AchE)-positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins lIb and IlIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen-stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.