巨核细胞形态学改变和特发性血小板减少性紫癜治疗的关系

目的探讨成人特发性血小板减少性紫癜(ITP)与骨髓巨核细胞的关系及其治疗时的形态学改变。方法回顾性分析52例ITP患者的临床资料、实验室检查及治疗效果,就骨髓巨核细胞的数量、退化变性、颗粒、细胞胞浆量与36例健康者进行定量比较分析。结果 ITP患者巨核细胞数明显增加,颗粒减少伴成熟障碍,胞浆量增多,退化变性增加,与对照组比较有显著性差异(P均<0.05)。治疗前血小板数与疗效无明显关系(P>0.05),但治疗前巨核细胞数增加或正常者效果明显优于巨核细胞数减少者(P均<0.05)。结论骨髓巨核细胞的形态学改变对诊断ITP有重要意义。肾上腺皮质激素治疗效果与巨核细胞数相关,巨核细胞数增加或正常者疗效较好。     

疏风凉血补肾方治疗慢性特发性血小板减少性紫癜临床研究

目的观察疏风凉血补肾方对慢性特发性血小板减少性紫癜患者(CITP)骨髓巨核细胞分化成熟及转化生长因子-β1(TGF-β1)的调节作用。方法将50例CITP患者随机分为中药组20例、联合组15例和西药组15例。中药组以疏风凉血补肾方治疗,联合组以疏风凉血补肾方加强的松治疗,西药组以强的松为主治疗,4周为1个疗程,连续治疗3个疗程,观察临床疗效及血小板计数、骨髓巨核细胞总数和构成比例、骨髓TGF-β1水平的变化。结果中药组良效率(55.0%,11/20)显著高于西药组(20.0%,3/15),与联合组(60.0%,9/15)相近;中药组治疗后血小板计数上升(P＜0.01),且优于西药组(P＜0.05),与联合组相似;中药组治疗后骨髓巨核细胞总数降低、产板巨核细胞比例上升(P＜0.01),与联合组相似,而西药组治疗前后无明显差异;中药组、联合组治疗后TGF-β1值降低(P＜0.05),且和西药组治疗后比较差异有统计学意义(P＜0.05)。结论疏风凉血补肾方能有效降低CITP患者TGF-β1水平,促进骨髓巨核细胞分化成熟,从而有效提高血小板计数。     

当归多糖在体外抑制HCMV感染所致巨核系细胞凋亡中的作用探讨

本研究探讨当归多糖（angelica polysaccharide,APS）在体外抑制HCMV感染致人巨核系细胞凋亡中的作用。HCMV AD169体外感染巨核系细胞CHRF-288-11,在病毒感染后第3天向试验体系中加入APS。用PCR扩增检测HCMV DNA,用形态学、DNA片段化、细胞表面标志检测APS对HCMV感染所致巨核系细胞凋亡的影响。结果表明：APS在一定程度上能抑制HCMV体外感染诱导的巨核系细胞CHRF-288-11凋亡,且呈剂量依赖性。感染的巨核系细胞CHRF-288-11中有hCMV IEA的表达,形态学和DNA片段化分析证实了细胞凋亡的存在,Annexin V／PI双染流式细胞仪分析显示受感染的CHRF细胞中随着加入APS剂量的减少,凋亡率呈上升趋势。结论：HC—MVAD169株在体外可直接感染巨核系细胞并降低其存活率;HCMVAD169株在体外感染巨核系细胞后可诱导其加重凋亡,凋亡率与时间呈正相关。在HCMVAD169株体外感染巨核系细胞后,向培养细胞中加入APS,可以提高受染细胞的存活率,表明APS对HCMV感染的巨核系细胞有一定的保护作用;APS在一定程度上能抑制HCMV体外感染诱导的巨核系细胞凋亡,且呈剂量依赖性。     

Transgenic,inducible RNAi in megakaryocytes and platelets in mice

Summary Background: RNA interference (RNAi) is a powerful tool for suppressing gene function. The tetracycline (tet)‐regulated expression system has recently been adapted to allow inducible RNAi in mice, however its efficiency in a particular cell type in vivo depends on a transgenic tet transactivator expression pattern and is often highly variable. Objective: We aimed to establish a transgenic strategy that allows efficient and inducible gene knockdown in particular hematopoietic lineages in mice. Methods and results: Using a tet‐regulated reporter gene strategy, we found that transgenic mice expressing the rtTA (tet‐on) transactivator under control of the cytomegalovirus (CMV) promoter (CMV‐rtTA mice) display inducible reporter gene expression with unusual and near‐complete efficiency in megakaryocytes and platelets. To test whether the CMV‐rtTA transgene can drive inducible and efficient gene knockdown within this lineage, we generated a novel mouse strain harboring a tet‐regulated short hairpin RNA (shRNA) targeting Bcl‐x_L, a pro‐survival Bcl‐2 family member known to be essential for maintaining platelet survival. Doxycycline treatment of adult mice carrying both transgenes induces shRNA expression, depletes Bcl‐x_L in megakaryocytes and triggers severe thrombocytopenia, whereas doxycycline withdrawal shuts off shRNA expression, normalizes Bcl‐x_L levels and restores platelet numbers. These effects are akin to those observed with drugs that target Bcl‐x_L, clearly demonstrating that this transgenic system allows efficient and inducible inhibition of genes in megakaryocytes and platelets. Conclusions: We have established a novel transgenic strategy for inducible gene knockdown in megakaryocytes and platelets that will be useful for characterizing genes involved in platelet production and function in adult mice.     

The mitogen-activated protein kinase signaling pathways: role in megakaryocyte differentiation

Summary.  Megakaryopoiesis is a process by which bone marrow progenitor cells develop into mature megakaryocytes (MKs), which in turn produce platelets required for normal hemostasis. The mitogen-activated protein kinases (MAPKs) family comprises four main groups of proteins: extracellular signal-related kinases (ERKs) (ERK1/2 or p44/p42), ERK5, p38MAPKs (α, β, γ, δ) and c-Jun amino-terminal kinases (JNKs) (JNK 1, 2, 3). These intracellular signaling pathways play a pivotal role in many essential cellular processes including proliferation and differentiation. The purpose of this review is to summarize our current knowledge on the role of MAPKs in MKs, specifically regarding differentiation in immortalized cell lines and primary MKs. A critical role of the MEK (MAPK kinase)-ERK1/2 pathway in MK development has been demonstrated although the details remain controversial. There is at present no functional evidence for a role of p38MAPKs whereas the role of JNKs and ERK5 in MK development is not known. Characterization of these molecular event cascades remains crucial for the understanding of the megakaryopoiesis process.     

骨髓巨核细胞数对小儿特发性血小板减少性紫癜预后的影响

目的探讨骨髓巨核细胞数对判断小儿特发性血小板减少性紫癜(ITP)预后的意义。方法计数ITP患 儿治疗前骨髓巨核细胞数,并将其分为巨核细胞数正常组和巨核细胞数增多组,给予相同的治疗方案,并随访1 .年。结果50例患儿治疗后,巨核细胞数正常组77.8％(21/27例)于2周内达完全反应,而巨核细胞数增多组仅 39.1％(9/23例)在2周内达完全反应。经统计学处理,两组有非常显著性差异(χ     

Bortezomib induces thrombocytopenia by the inhibition of proplatelet formation of megakaryocytes

Bortezomib is a potent proteasome inhibitor that has been extensively used to treat multiple myeloma. One of the most common grade 3 adverse events is cyclic thrombocytopenia. In this study, we studied the mechanism by which bortezomib induces thrombocytopenia in a mouse model. After the intravenous administration of bortezomib (2.5 mg/kg) via tail vein, platelet counts significantly decreased on days 2–4 and recovered to the normal range on day 6. Bortezomib (2.5 mg/kg) injected into mice in vivo did not affect colony‐forming unit‐megakaryocytes (CFU‐Mk) or megakaryocytes in the bone marrow. However, proplatelet formation (PPF) significantly decreased on days 2 and 4, after bortezomib administration to mice. Meanwhile, CFU‐Mk formation and the ploidy distribution of cultured megakaryocytes in vitro were not affected by bortezomib used at concentrations of ≤1 ng/mL. The PPF of megakaryocytes in vitro significantly decreased with 0.1, 1, 10, and 100 ng/mL bortezomib. Considering the bortezomib concentration in clinical studies, these data strongly suggest that decreased PPF activity induces thrombocytopenia. To elucidate the mechanism behind decreased PPF, Western blot was performed. Activated Rho expression increased after the incubation of murine platelets with bortezomib. Decreased PPF activity was eliminated by the addition of Y27632, a Rho kinase inhibitor, in vitro. Given that the Rho/Rho kinase pathway is a negative regulator of PPF, bortezomib increases activated Rho, inducing decreased PPF, which results in decreased platelet count.     

早孕小鼠血小板减少机制的组织学研究

应用昆明小鼠对其妊娠早期、假孕、脾摘除妊娠早期外周血血小板计数的变化进行观察。结果表明，正常小鼠和脾摘除小鼠妊娠早期外周血中血小板计数明显减少，约１周后恢复正常。假孕小鼠无此反应。血小板计数减少与生殖管道内的胚卵数呈负相关。脾的组织学观察表明，妊娠第４天小鼠脾红髓内巨核细胞数较假孕第４天明显减少。提示早孕小鼠血小板计数减少可能与生殖道内的胚卵数、血小板激活因子及脾内巨核细胞数有关。