Proportion Of Megakaryocytes Research Articles

Establishment of a platelet production model by bone marrow cavity transplantation of mouse primary megakaryocytes

目的利用小鼠原代巨核细胞骨髓腔移植构建血小板产生模型，为研究巨核细胞功能及血小板产生调控机制提供工具。方法通过磁珠富集绿色荧光蛋白（Green Fluorescent Proteins, GFP）转基因供体小鼠骨髓原代巨核细胞，将其移植入经半致死剂量辐照的受体小鼠骨髓腔，建立原代巨核细胞骨髓腔内血小板生成模型。通过免疫荧光染色、流式细胞术等方法检测受体小鼠中供体来源巨核细胞和血小板形态、大小等指标。结果磁珠富集可将巨核细胞在骨髓细胞悬液中的比例提高40～50倍。供体来源巨核细胞能够在受体小鼠骨髓腔成功定植并正常产生血小板，其产生的子代血小板具有与受体自身来源血小板相似的形态、大小及CD41、CD42d、CD61等表面标志分子表达水平。结论通过磁珠富集与骨髓腔注射可成功构建小鼠原代巨核细胞移植模型，该模型能够客观反映巨核细胞在骨髓内产生血小板的能力。

Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms

AimsMegakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of...

Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias.

Abnormal organization of platelet microtubules is associated with abnormal platelet formation in hereditary macrothrombocytopenias such as the gray platelet syndrome, May-Hegglin anomaly, and Epstein's syndrome, and that of the Wistar Furth rat, suggesting that aberrant microtubule organization may contribute to defective platelet formation in these clinical entities. Here, we examined the consequence of microtubule disruption on the organization of megakaryocyte cytoplasmic organelles using the microtubule depolymerizing agent, vincristine (VCR). Wistar rat bone marrow was fixed and processed for transmission electron microscopy after VCR administration alone, after 5-fluorouracil (5-FU) administration alone, or after 5-FU followed by intravenous injection of 0.1-1.0 mg/kg VCR for intervals of 30 min to 8 hr. 5-FU was given to increase megakaryocyte frequency to facilitate ultrastructural evaluations. VCR alone or in combination with 5-FU caused formation of large membrane complexes in the cytoplasm of Wistar rat megakaryocytes at all dosages studied, identical to those found in megakaryocytes of human hereditary macrothrombocytopenias and the Wistar Furth rat. The proportion of megakaryocytes with these large membrane complexes increased with time after 5-FU and VCR, and was maximal (approximately two-thirds of megakaryocytes) at VCR dosages of 0.75-1.0 mg/kg. The majority of megakaryocytes displayed other abnormalities, including blebbing of plasma membranes, an increased number of dense compartments, dilated demarcation membrane (DMS) channels, which contained dense material immunocytochemically identified as secreted alpha-granule proteins, and an increased incidence of emperipolesis. Rats administered 5-FU alone did not demonstrate these abnormalities, with the exception of an increase in dense compartments. Platelets from rats treated with VCR alone or 5-FU and VCR also showed abnormalities including membrane complexes, rounded shape, formation of tubulin paracrystals, development of membrane blebs, and the presence of proteinaceous material within the cisternae of the surface-connected canalicular system (SCCS). The membrane complexes in platelets of 5-FU-, VCR-treated Wistar rats as well as untreated Wistar Furth rats were composed of elements of both the SCCS and dense tubular system; membrane complexes in megakaryocytes of 5-FU-, VCR-treated rats were composed of both DMS and smooth endoplasmic reticulum. We conclude that intact microtubules play a major role in the organization of the megakaryocyte DMS and may contribute to the stability of megakaryocyte alpha-granules.

Stimulation of megakaryocytopoiesis in mice by human recombinant interleukin-6

The in vivo effects of purified human recombinant interleukin-6 (IL-6) on murine megakaryocytopoiesis were examined. IL-6 was administered subcutaneously to Swiss Webster mice, followed by evaluation of bone marrow megakaryocyte ploidy, size and frequency, and median platelet volume 24, 48, and 72 hours after the initiation of IL-6 administration. In addition, bone marrow megakaryocyte morphology was examined using electron microscopy at 72 hours. IL-6 (10,000 U per subcutaneous injection) was administered three times during the first 24 hours, three times during the second 24 hours, and twice during the last 24-hour period. IL-6 bioactivity (10 U/ng) was determined using the IL-6-dependent murine hybridoma cell line B9. Megakaryocyte ploidy distribution, measured by two-color flow cytometry, demonstrated a shift in the modal ploidy class from 16N to 32N and a significant increase in the relative frequency of 64N megakaryocytes 48 and 72 hours (but not 24 hours) after initiation of IL-6 administration (cumulative doses of 60,000 and 80,000 U at 48 and 72 hours, respectively). In addition, ploidy levels were increased in animals that received a cumulative IL-6 dose of only 40,000 U (evaluated after 72 hours). The size of recognizable bone marrow megakaryocytes, determined by the cross-sectional areas of plastic embedded bone marrow megakaryocytes, was increased at the 48-hour (60,000 U IL-6) and 72- hour (80,000 U IL-6) time points. Megakaryocyte frequency, measured by flow cytometry, was unaffected at all time points and doses of IL-6. Median platelet volume, measured by electrical impedance, was not consistently altered by administration of IL-6. Electron microscopic examination of bone marrow megakaryocytes showed an increase in the proportion of megakaryocytes with a wide, peripheral, organelle- deficient zone from 20% +/- 9% (SD) in control animals to 50% +/- 7% (SD) (P less than .02) in animals that received IL-6. No changes were observed in the distribution of the demarcation membranes. IL-6 is a potent stimulator of murine megakaryocytopoiesis, in vivo, and appears to act early in megakaryocyte differentiation.

Stimulation of Megakaryocytopoiesis in Mice by Human Recombinant Interleukin-6

The in vivo effects of purified human recombinant interleukin-6 (IL-6) on murine megakaryocytopoiesis were examined. IL-6 was administered subcutaneously to Swiss Webster mice, followed by evaluation of bone marrow megakaryocyte ploidy, size and frequency, and median platelet volume 24, 48, and 72 hours after the initiation of IL-6 administration. In addition, bone marrow megakaryocyte morphology was examined using electron microscopy at 72 hours. IL-6 (10,000 U per subcutaneous injection) was administered three times during the first 24 hours, three times during the second 24 hours, and twice during the last 24-hour period. IL-6 bioactivity (10 U/ng) was determined using the IL-6-dependent murine hybridoma cell line B9. Megakaryocyte ploidy distribution, measured by two-color flow cytometry, demonstrated a shift in the modal ploidy class from 16N to 32N and a significant increase in the relative frequency of 64N megakaryocytes 48 and 72 hours (but not 24 hours) after initiation of IL-6 administration (cumulative doses of 60,000 and 80,000 U at 48 and 72 hours, respectively). In addition, ploidy levels were increased in animals that received a cumulative IL-6 dose of only 40,000 U (evaluated after 72 hours). The size of recognizable bone marrow megakaryocytes, determined by the cross-sectional areas of plastic embedded bone marrow megakaryocytes, was increased at the 48-hour (60,000 U IL-6) and 72-hour (80,000 U IL-6) time points. Megakaryocyte frequency, measured by flow cytometry, was unaffected at all time points and doses of IL-6. Median platelet volume, measured by electrical impedance, was not consistently altered by administration of IL-6. Electron microscopic examination of bone marrow megakaryocytes showed an increase in the proportion of megakaryocytes with a wide, peripheral, organelle-deficient zone from 20% +/- 9% (SD) in control animals to 50% +/- 7% (SD) (P less than .02) in animals that received IL-6. No changes were observed in the distribution of the demarcation membranes. IL-6 is a potent stimulator of murine megakaryocytopoiesis, in vivo, and appears to act early in megakaryocyte differentiation.