血小板相关抗体、巨核细胞数量对免疫性与非免疫性血小板减少患者的诊断意义

目的比较骨髓巨核细胞数量、血小板相关抗体在免疫性血小板减少和非免疫性血小板减少患者中的不同,探讨巨核细胞数量、血小板相关抗体在血小板减少症诊断中的意义。方法观察132例ITP患者及48例非ITP患者的骨髓巨核细胞数量、血小板相关抗体水平,进行统计分析。结果 132例ITP患者中巨核细胞数量正常者35例,增多者78例,77例ITP患者中血小板抗体阳性58例。结论由于血小板抗体生成使ITP患者巨核细胞成熟障碍,因此血小板生成减少,骨髓巨核细胞数量在免疫性血小板减少的诊断中是必要的检查,血小板抗体与巨核细胞数量相关。     

重组人白细胞介素11对两种血小板减少症患儿骨髓巨核系祖细胞的作用

目的研究重组人白细胞介素11（recombinant human interleukin 11，rhIL-11）对慢性特发性血小板减少性紫癜（chronic idiopathic thrombocytopenic purpura，CITP）与再生障碍性贫血（aplastic anemia，AA）患儿骨髓巨核系祖细胞（megakaryocyte progenitor）体外生长的影响。方法收集17例CITP患儿及14例AA患儿骨髓，对照组为骨髓检查三系造血功能正常、排除血液系统疾病儿童10例。无血清培养各组骨髓单个核细胞（mononuclear cells，MNC），培养体系中包含血小板生成素（TPO）、可溶性补体结合（SCF），IL-3，rhIL-11，或TPO，SCF，IL-3。培养第14天，用流式细胞仪测定CD41^＋细胞率，CD41结合的碱性磷酸酶抗碱性磷酸酶（alkaline phosphatase—anti—alkaline phosphatase，APAAP）桥联酶标技术鉴定半固体培养的巨核细胞集落形成单位（megakaryocyte colony forming unit，CFU—MK）和巨核细胞爆式集落形成单位（megakaryocyte burst forming unit，BFU—MK）。结果rhIL-11联合TPO等细胞因子，可促进对照组、CITP组CFU—MK和BFU—MK生成（P〈0．01），提高CD41^＋细胞率（P〈0．01）；但对AA组，CFU—MK和CD41^＋细胞率无明显的作用（P〉0．05）。结论rhIL-11联合TPO等细胞因子，可促进CITP患儿骨髓巨核系祖细胞在体外无血清培养条件下的增殖，提示rhIL-11具有治疗CITP的潜在价值，但对AA患儿的骨髓巨核系祖细胞生成并无明显的效果。     

Uncaria tomentosa stimulates the proliferation of myeloid progenitor cells

Ethnopharmacological relevance

The Asháninkas, indigenous people of Peru, use cat's claw (Uncaria tomentosa) to restore health. Uncaria tomentosa has antioxidant activity and works as an agent to repair DNA damage. It causes different effects on cell proliferation depending on the cell type involved; specifically, it can stimulate the proliferation of myeloid progenitors and cause apoptosis of neoplastic cells. Neutropenia is the most common collateral effect of chemotherapy. For patients undergoing cancer treatment, the administration of a drug that stimulates the proliferation of healthy hematopoietic tissue cells is very desirable.It is important to assess the acute effects of Uncaria tomentosa on granulocyte-macrophage colony-forming cells (CFU-GM) and in the recovery of neutrophils after chemotherapy-induced neutropenia, by establishing the correlation with filgrastim (rhG-CSF) treatment to evaluate its possible use in clinical oncology.

Materials and methods

The in vivo assay was performed in ifosfamide-treated mice receiving oral doses of 5 and 15 mg of Uncaria tomentosa and intraperitoneal doses of 3 and 9 μg of filgrastim, respectively, for four days. Colony-forming cell (CFC) assays were performed with human hematopoietic stem/precursor cells (hHSPCs) obtained from umbilical cord blood (UCB).

Results

Bioassays showed that treatment with Uncaria tomentosa significantly increased the neutrophil count, and a potency of 85.2% was calculated in relation to filgrastim at the corresponding doses tested. An in vitro CFC assay showed an increase in CFU-GM size and mixed colonies (CFU-GEMM) size at the final concentrations of 100 and 200 μg extract/mL.

Conclusions

At the tested doses, Uncaria tomentosa had a positive effect on myeloid progenitor number and is promising for use with chemotherapy to minimize the adverse effects of this treatment. These results support the belief of the Asháninkas, who have classified Uncaria tomentosa as a ‘powerful plant’.     

Megakaryocytic differentiation is accompanied by a reduction in cell migratory potential

Megakaryocytes (MKs) have been found in the peripheral circulation, suggesting that they can migrate out of the bone marrow. In order to evaluate if megakaryocytic differentiation confers a migratory phenotype, we investigated this property in the haematopoietic cell lines MO7e and UT-7/mpl and in CD34+ progenitor cells before and after induction of differentiation by thrombopoietin (TPO). Migration was studied using a bicompartmental culture system in the presence or absence of a bone marrow endothelial cell monolayer. Preincubation with TPO led to a significant reduction in stromal cell-derived factor-1 (SDF-1)-induced migration of MO7e cells (0.7% +/- 0.08% for TPO-treated vs. 2.6% +/- 0.3% for controls P < 0.05). A similar decreased migratory response was seen with UT-7/mpl cells (7.4% +/- 0.4% for TPO-treated vs. 11.1% +/- 0.01% for controls, P<0.05), although these cells did not migrate in response to SDF-1. CD34+ cells partially differentiated with TPO showed decreased migration following further TPO-induced maturation (13.9% +/- 1.8% for TPO-treated vs. 24.1% +/- 1.8% for untreated, P < 0.05). This reduction was more pronounced in the large MK (> or = 4n) fraction. These results demonstrate that megakaryocytic differentiation is accompanied by a partial suppression of the haematopoietic cell migratory phenotype.     

Investigation of megakaryocyte apoptosis in children with acute and chronic idiopathic thrombocytopenic purpura

OBJECTIVE: Although the platelet destruction shows a primary role in the thrombocytopenia of idiopathic thrombocytopenic purpura (ITP), it has been demonstrated that impaired platelet production may also contribute to the severity of thrombocytopenia in ITP. The present study examined megakaryocyte apoptosis in bone marrow aspirates of children with acute and chronic ITP and investigated the role of megakaryocyte apoptosis in ITP pathophysiology. METHODS: Thirteen children diagnosed with acute ITP and eight children diagnosed with chronic ITP comprised the study group. Ten children, who were hospitalized for scoliosis operation but healthy otherwise, comprised the control group. In all children, megakaryocytes were isolated from the same amount of bone marrow aspirate samples using MACS CD61 MicroBeads (Miltenyl Biotec, Auburn, CA, USA). Megakaryocyte apoptosis was studied with transferase-mediated d-UTP-bitin nick end-labeling method. RESULTS: Isolated megakaryocyte counts did not differ significantly between acute ITP, chronic ITP and control groups. The percentage of apoptotic megakaryocytes did not differ significantly between acute ITP group and control group and between chronic ITP group and control group. The percentage of apoptotic megakaryocytes in patients with chronic ITP was significantly lower than the patients with acute ITP. There was no correlation between the percentage of apoptotic megakaryocytes and platelet counts of the cases. CONCLUSIONS: Increased megakaryocytic apoptosis does not play a role in the pathogenesis of dysmegakaryopoiesis and impaired platelet production in children with ITP. Decreased megakaryocyte apoptosis in cases with chronic ITP may be due to suppression of megakaryocyte maturation, as the terminal phase of the megakaryocyte lifespan is characterized by the onset of apoptosis.     

脐血和骨髓来源的CD34^＋细胞体外扩增巨核祖细胞差异的研究

本研究探讨脐血（CB）和骨髓（BM）来源的CD34^＋细胞体外扩增巨核祖细胞的差异。采用Ficoll—Hypaque分离法分离人CB及BM单个核细胞，免疫磁珠法制备CD34^＋细胞，在含血小板生成素（TPO）、TPO＋白介素11（IL—11）或TPO＋IL11＋肝素的无血清液体培养体系中培养14天。流式细胞术检测扩增产物（CD34^＋、CD41a^＋及CD34^＋CD41a^＋细胞）免疫表型、巨核细胞凋亡率及DNA含量，并以集落形成单位测定法进行粒巨-噬细胞集落形成单位（CFU—GM）、红系爆式集落形成单位（BFU—E）及巨核细胞集落形成单位（CFU—Mk）计数。结果表明：14天培养中，CB来源细胞在总细胞数、CD41a^＋及CD34^＋CD41a^＋细胞扩增倍数上均高于BM（P均〈0．05）。0天CB及BM来源CD34^＋细胞在CFU—GM、BFU—E及总的CFU—Mk的形成能力上无显著性差异（P均〉0．05），但CB来源CD34^＋细胞形成的CFU—Mk以大集落为主，其数量高于BM（P〈0．05）；在培养7、10和14天，CB及BM来源细胞CFU—GM扩增倍数无显著性差异（P均〉0．05），但CB来源细胞的BFU—E及总的CFU—Mk扩增倍数均高于BM（P均〈0．05）。14天培养中CB和BM来源巨核细胞的凋亡率无显著性差异（P均〉0．05）。DNA含量检测发现，14天培养中CB来源巨核细胞始终以2N细胞为主（比例〉90％），而BM来源巨核细胞随着培养时间延长，4N、8N及以上倍体巨核细胞比例逐渐增加。结论：CB来源CD34^＋细胞体外扩增巨核祖细胞能力高于BM，它可能是巨核祖细胞体外扩增较好的来源。     

Regulation of platelet biogenesis: insights from the May–Hegglin anomaly and other MYH9-related disorders

Summary.  Megakaryocyte (MK) maturation culminates in release of blood platelets through proplatelet extensions. MKs presumably delay elaborating proplatelets until synthesis of platelet constituents is complete. Recent insights from investigation of a classic human congenital macrothrombocytopenia, the May–Hegglin anomaly, and related MYH9 -associated disorders shed new light on underlying mechanisms. The findings reviewed in this article implicate myosin IIA, the non-muscle myosin heavy chain product of the MYH9 gene, in restraining proplatelet formation until MKs achieve terminal maturity. Loss of myosin IIA function, through dominant inhibitory mutations in humans, targeted gene disruption in mice, or manipulation of cultured MKs, seems to accelerate proplatelet formation. The resulting process is inefficient and produces platelets that vary widely in size, shape and content. Several lines of evidence suggest that the Rho-ROCK-myosin light chain pathway restrains proplatelet formation through myosin IIA. These findings illustrate that mammalian thrombopoiesis is complex and subject to both positive and negative regulation.     

CD226 is expressed on the megakaryocytic lineage from hematopoietic stem cells/progenitor cells and involved in its polyploidization

OBJECTIVES: In this study, we examined the expression of CD226 on megakaryocytic, granulocytic and erythroid lineage from hematopoietic stem cells/progenitor cells in adult and fetus and its potential role in megakaryocytic maturation. METHODS: CD34(+) cells from adult and fetus were induced to differentiate toward the megakaryocytic lineage by thrombopoietin (TPO) and the granulocytic lineage by granulocyte colony-stimulating factor (G-CSF), respectively. Mononuclear cells from fetal liver and CD34(+) cells from adult were induced to differentiate toward erythroid-lineage by erythropoiesis (EPO). We investigated the expression of CD226 and lymphocyte function associated antigen-1 (LFA-1) (CD11a) during hemopoiesis. We also studied the effect of CD226 monoclonal antibody (MoAb) and LFA-1 MoAb on megakaryocyte with antibody cross-liking technique. RESULTS: CD34(+) cells from adult and fetus and TPO-induced CD41(+) cells all expressed CD226 molecule. CD226 was not expressed on erythroid progenitor cells and erythroblasts and most cells of granulocytic lineage although G-CSF induced a significant increase of the expression of CD226 on CD34(+) cells in early period of time. CD226 MoAb acts on megakaryocytes by inducing intracellular calcium mobilization. The expression of LFA-1 decreased significantly at late stage of differentiation and maturation of fetal megakaryocytes whereas the expression of LFA-1 on adult megakaryocytes retained at a high level. CD226 MoAb in combination with LFA-1 MoAb shifted the ploidy of generated megakaryocytes from adult-derived CD34(+) cells to higher classes significantly although CD226 and LFA-1 MoAb slightly increased the ploidy of the generated megakaryocytes individually. CD226 MoAb or LFA-1 MoAb or CD226 MoAb plus LFA-1 MoAbs did not increase the ploidy of the generated megakaryocytes from fetus-derived CD34(+) cells. CONCLUSION: CD226 molecules play an important role in maturation of the megakaryocytes in combination with LFA-1.     

The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high-dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony-forming units-megakaryocyte; CFU-Mk) with high- (> 20 cells/colony) and low-proliferative capacity (5-20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL-3) or IL-3 + IL-6 + IL-11 + Flt3-ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell-derived factor 1 (SDF-1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF-1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL-3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.