三氧化二锑诱导急性早幼粒细胞白血病细胞凋亡的研究

目的 研究锑剂三氧化二锑（Sb2O3)对早幼粒细胞白血病细胞株NB4凋亡的诱导作用，以寻求早幼粒细胞白血病治疗的新方法。方法 采用细胞生长曲线，形态学及硝基四氮唑蓝（NBT）还原试验，判定NB4细胞的生长，分化及功能。采用细胞周期分析和DNA电泳研究细胞凋亡。结果 Sb2O3能诱导早幼粒白血病细胞凋亡，且具有时间，剂量依赖性。结论 Sb2O3能有效地诱导早幼粒白血病细胞凋亡，提示锑剂诱导细胞半亡的疗法，有望成为临床治疗早幼粒细胞白血病的新方法。     

Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). With the application of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), APL becomes one of best prognosis of leukemia. However, ATRA and ATO are not effective against all APLs. Therefore, a new strategy for APL treatment is necessary. Here, we investigated whether lithium chloride (LiCl), a drug used for the treatment of mental illness, could promote apoptosis in human leukemia NB4 cells. We observed that treatment with LiCl significantly accelerated apoptosis in NB4 cells and led to cell cycle arrest at G2/M phase. Moreover, LiCl significantly increased the level of Ser9-phosphorylated glycogen synthase kinase 3β(p-GSK-3β), and decreased the level of Akt1 protein in a dose-dependent manner. In addition, LiCl inhibition of c-Myc also enhanced cell death with a concomitant increase in β-catnin. Taken together, these findings demonstrated that LiCl promoted apoptosis in NB4 cells through the Akt signaling pathway and that G2/M phase arrest was induced by increase of p-GSK-3β(S9).     

IL-1对维甲酸诱致高白细胞症的影响

使用生物活性检测法检测ATRA治疗前后APL患者血清中IL-1活性的动态变化,并分析IL-1活性与外周血白细胞数变化的相关性。观察经ATRA作用后不同时相NB4细胞、巨噬细胞和血管内皮细胞上清液中IL-1活性的动态变化,并以RT-PCR法检测NB4细胞中IL-1 mRNA表达的变化。结果表明,患者外周血白细胞数及粒细胞数与血清中IL-1活性的变化相关;ATRA作用后,NB4细胞、巨噬细胞和血管内皮细胞上清液中IL-1活性均呈时间依赖性增高;ATRA可上调NB4细胞中IL-1的表达。结论:ATRA治疗APL过程中,多种细胞来源的IL-1可能参与了高白细胞症的发生。     

Selective up-regulation of phospholipase C-beta2 during granulocytic differentiation of normal and leukemic hematopoietic progenitors

In this study, we have investigated the expression of phospholipase C-beta2 during the course of granulocytic differentiation of normal and malignant progenitors. As a model system, we used the NB4 cell line, a reliable in vitro model for the study of acute promyelocytic leukemia (APL), a variety of acute myeloid leukemia (AML) that responds to pharmacological doses of all trans-retinoic acid (ATRA) by differentiating in a neutrophil-like manner. We found that PLC-beta2, virtually absent in untreated NB4 cells, was strongly up-regulated after ATRA-induced granulocytic differentiation. Remarkably, using primary blasts purified from bone marrow of patients affected by APL successfully induced to remission by treatment with ATRA, we showed a striking correlation between the amount of PLC-beta2 expression and the responsiveness of APL blasts to the differentiative activity of ATRA. An increase of PLC-beta2 expression also characterized the cytokine-induced granulocytic differentiation of CD34+ normal hematopoietic progenitors. Taken together, these data show that PLC-beta2 represents a sensitive and reliable marker of neutrophil maturation of normal and malignant myeloid progenitors. Moreover, PLC-beta2 levels can predict the in vivo responsiveness to ATRA of APL patients.     

免疫荧光动态监测急性早幼粒细胞白血病患者PML-RARα融合蛋白变化

背景：实时定量RT-PCR只能间接地反映融合基因表达量的变化,并不能在细胞原位直观反映急性早幼粒细胞白血病患者细胞内PML-RARα融合蛋白在全反式维甲酸治疗前后的相对表达量。 目的：探讨免疫荧光技术检测融合蛋白PML-RARα表达的动态变化在判断急性早幼粒细胞白血病的全反式维甲酸早期治疗的效果。 方法：应用免疫荧光技术检测全反式维甲酸干预的急性早幼粒细胞白血病经典细胞株NB4细胞或来源于急性早幼粒细胞白血病患者的NB4细胞PML-RARα蛋白的表达,以实时定量RT-PCR检测细胞PML-RARα mRNA的表达作为对照,以评价免疫荧光技术的可靠性。 结果与结论：免疫荧光染色显示,无论是体外培养的NB4细胞,还是患者骨髓内的NB4细胞,在全反式维甲酸干预前细胞内均可见大量红色的融合蛋白PML-RARα,大小不均一、形状不规则,全反式维甲酸干预后,NB4细胞内红色的融合蛋白PML-RARα形状逐渐规则,弥散状态变为散在分布,且随着全反式维甲酸干预时间的延长,原有的弥散状态逐渐消失。实时定量RT-PCR测得的PML-RARα mRNA的变化趋势与免疫荧光相一致。说明免疫荧光技术检测PML-RARα能够更为直观地判断全反式维甲酸的治疗效果。 关键词：免疫荧光;实时定量RT-PCR;PML-RARα;急性早幼粒细胞白血病;NB4细胞;U937细胞 doi:10.3969/j.issn.1673-8225.2012.10.036     

NLS-RARα Inhibits the Effects of All-trans Retinoic Acid on NB4 Cells by Interacting with P38α MAPK

Nuclear localization signal retinoic acid receptor alpha(NLS-RARα), which forms from the cleavage of promyelocytic leukemia-retinoic acid receptor alpha(PML-RARα) protein by neutrophil elastase(NE), possesses an important role in the occurrence and development of acute promyelocytic leukemia(APL). However, the potential mechanism underlying the effects of NLS-RARα on APL is still not entirely clear. Here, we investigated the effects of NLS-RARα on APL NB4 cells and its mechanism. We found that all-trans retinoic acid(ATRA) could promote differentiation while inhibit proliferation of APL NB4 cells via upregulating the expression of phosphorylated p38α mitogen-activated protein kinase(p-p38α MAPK). We also found that NLS-RARα could inhibit differentiation while accelerate proliferation of NB4 cells via downregulating the expression of p-p38α protein in the presence of ATRA. Furthermore, immunofluorescence and co-immunoprecipitation assays confirmed NLS-RARα interacted with p38α protein directly. Finally, application of PD169316, an inhibitor of p38α protein, suggested that recruitment p38α-combinded NLS-RARα by ATRA eventually caused activation of p38α protein. In summary, our study demonstrated that ATRA cound promote differentiation while inhibit proliferation of APL NB4 cells via activating p38α protein after recruiting p38α-combinded NLS-RARα, while NLS-RARα could inhibit the effects of ATRA in the process.     

干扰TGF-βⅡ型受体表达对NB4细胞增殖、分化及凋亡的影响

目的:研究干扰TGF-βⅡ型受体(TβRⅡ)表达对人急性早幼粒细胞白血病NB4细胞生长、分化及凋亡的影响。方法:应用慢病毒介导RNA干扰技术,下调NB4细胞TβRⅡ基因的表达,获得TβRⅡ-shRNA NB4细胞,CCK-8法检测细胞的活力,流式细胞术检测CD11b表达,并用瑞氏-吉姆萨染色法检测全反式维甲酸对细胞分化的影响; Annexin V-FITC/PI双荧光标记法及AO/EB染色观察三氧化二砷对细胞凋亡的影响。结果:TβRⅡ-shRNA NB4细胞的活力高于NB4细胞。采用不同浓度(0. 01、0. 02、0. 04、0. 08和0. 1μmol/L)的全反式维甲酸进行96 h的孵育后,2组细胞均出现分化现象(CD11b表达增加),并呈剂量依赖性,但TβRⅡ-shRNA NB4细胞的分化率低于NB4细胞。不同浓度(2、4和8μmol/L)的三氧化二砷孵育24 h后,2组细胞均出现凋亡现象(AO/EB染色),呈剂量依赖性,但TβRⅡ-shRNA NB4细胞凋亡率显著低于NB4细胞,其中用8μmol/L三氧化二砷孵育TβRⅡ-shRNA NB4细胞和NB4细胞24 h,凋亡率分别是(49. 15±2. 05)%和(66. 85±2. 41)%(P 0. 01)。结论:下调TβRII可以促进NB4细胞的生长,部分拮抗全反式维甲酸诱导的细胞分化及三氧化二砷诱导的细胞凋亡。     

丹参酮Ⅱa对急性髓系白血病NB4细胞增殖和凋亡的影响及其机制

目的观察丹参酮Ⅱa对急性髓系白血病细胞增殖和凋亡的影响,并初步分析其作用机制.方法人急性髓系白血病细胞NB4分别用不同浓度的丹参酮Ⅱa处理,11.2μmol/L柔红霉素作为阳性对照,未给予药物作为阴性对照.观察NB4细胞增殖、细胞周期、凋亡及相关通路蛋白的变化.结果丹参酮Ⅱa可明显抑制NB4细胞增殖,诱导细胞周期发生G1期阻滞,促进其凋亡,具有浓度依赖性(P<0.05).丹参酮Ⅱa可剂量性下调p-PI3K/PI3K、p-AKT/AKT及m-TOR的表达(P<0.05).结论丹参酮Ⅱa可抑制NB4细胞增殖,诱导细胞周期发生G1期阻滞,促进其凋亡,可能与抑制PI3K/AKT/m-TOR信号通路有关.     

初发急性髓性白血病患者外周血中高表达TAL1基因及其意义

目的:分析调控造血分化的重要转录因子TAL1在急性髓性白血病(AML)细胞株及原代AML细胞中的表达特点。方法:利用real-time PCR法分别检测47例初发急性髓性白血病患者外周血单个核细胞以及急性白血病细胞株(Jurkat、CCRF-CEM、HL-60和NB4细胞株)中TAL1 mRNA的水平,并以12例健康志愿者外周血样本作为对照组。结果:TAL1 mRNA在AML细胞株(HL-60和NB4)、T细胞急性淋巴细胞白血病(T-ALL)细胞株(Jurkat和CCRF-CEM)和原代AML细胞中的水平均显著高于健康对照组(P0.05)。各AML亚型中TAL1的mRNA表达水平均较对照组显著升高,并以AML-M1和AML-M5 2个亚型升高最为明显(P0.05)。结论:AML细胞中TAL1异常高表达可能与其影响粒系的分化发育有关,其能否作为AML发病相关分子标志物有待进一步研究。     

Binding and internalization of leptin by porcine choroid plexus cells in culture

Leptin is an adipocyte derived hormone with profound behavioural and metabolic effects exerted by both central and peripheral sites of action. One of its targets in the central nervous system appears to be the epithelial cells of the choroid plexus where leptin receptor (OB-R) expression is particularly high. The most abundant receptor subtype at this site is OB-Ra which is truncated at its intracellular part and has been suggested to serve functions such as leptin transport or clearance. The choroid plexus may thus be a site where receptor mediated exchange of leptin between cerebrospinal fluid and blood takes place. The study here shows that porcine plexus epithelia preserve their ability of OB-R expression when grown in culture. In addition, our experiments suggest that leptin is rapidly internalized upon binding to these cells supporting the view of an OB-R mediated transport of leptin across the choroid plexus.     

MicroRNA-26a的表达对急性髓系白血病细胞增殖的影响

目的观察构建has-miR-26a重组慢病毒的表达载体对急性髓系白血病细胞增殖能力的影响。方法构建人MicroRNA-26a慢病毒表达载体(LV-miR-26a-GFP)并感染人急性髓系白血病细胞系NB4;分为未感染组、感染组(感染LV-miR-26a-GFP)以及阴性对照组(感染LV-GFP);用real-time RT-PCR检测miR-26a在NB4细胞内的表达水平,Western blot检测PTEN的表达;用CCK-8法绘制细胞的生长曲线和软琼脂克隆形成实验检测细胞的增殖。结果成功构建miR-26a重组慢病毒的表达载体,感染白血病细胞NB4后,能够成功过表达miR-26a。生长曲线的结果显示与感染LV-GFP阴性对照组和未感染组相比,LV-miR-26a-GFP感染组细胞的增殖速度显著增加(P<0.01),软琼脂克隆形成实验证实了感染LV-miR-26a-GFP的NB4细胞的克隆形成能力(GFP+克隆数为75+10)与阴性对照组(感染LV-GFP)相比(GFP+克隆数为26+5)显著增强(P<0.05)。在NB4细胞中过表达miR-26a后,其靶基因PTEN的表达明显被抑制。结论 MicroRNA-26a的表达可增强急性髓系白血病细胞的增殖能力,促进白血病细胞的增殖。     

In vitro exposure of acute promyelocytic leukemia cells to arsenic trioxide (As2O3) induces the solitary expression of CD66c (NCA-50/90), a member of the CEA family

Arsenic trioxide (As2O3) is a useful drug for the treatment of acute promyelocytic leukemia (APL), acting through a complex mechanism involving the induction of apoptosis. We investigated by flow cytometry whether in vitro treatment of APL leukemic cells with As2O3 determined specific surface membrane changes. Twelve APL bone marrow aspirates were analyzed following 7 days of in vitro treatment with As2O3 (0.25, 0.5 and 2.5 microM) with regard to the expression of a series of differentiation antigens. Twelve acute myeloid leukemia (AML) samples of non-APL morphotype were analyzed as controls. Exposure of APL as well as non-APL samples to any concentration of As2O3 did not affect the expression of beta2 integrins (CD11a and CD11b), CD45 isoforms (RA, RB and R0), CD44/H-CAM, CD33 and the CEA-related antigen family members CD66ade and CD66b, thus failing to disclose any maturating effect. Of interest, in all APL samples (but not in AML) every tested dose of As2O3 determined a dramatic upregulation of CD66c display; intermediate concentration (0.5 microM) of As2O3 increased the median percentage of CD66c+ cells from 5% in control cultures (25th-75th percentile 2-12%) to 80% in drug-exposed cultures (25th-75th percentile 58-90%) (P<0.001). The induction of solitary expression of CD66c is a new finding which demonstrates As2O3 capability of generating phenotypic changes absolutely restricted to APL cells Moreover, these results provide experimental basis for considering the involvement of the newly described CD66 signalling pathway in As2O3-driven programmed cell death.     

Leptin--the fat controller

Obesity is a common health disorder in humans and is inherited genetically. Though several theories have been proposed in the past to understand the mechanisms underlying the control of obesity, the recent discovery of leptin (OB) has made the obesity research interesting. OB, a product of ob gene is a 16 KD protein, secreted by the adipocytes. It acts through its receptor (OB-R), which is a product of db gene. ob and OB-R in conjunction with neuropeptide Y, melanocyte stimulating hormone and melanocortin-4 receptor have been found to control adiposity. Though several issues pertaining to ob need to be addressed, it is anticipated that future treatment of obesity may depend on our understanding of the action(s) of leptin and its associated molecules and receptors.     

Thioredoxin-1 inhibitor PX-12 induces human acute myeloid leukemia cell apoptosis and enhances the sensitivity of cells to arsenic trioxide

Thioredoxin-1 (Trx-1), an important redox regulatory factor, plays a significant role in drug-induced apoptosis. Here we investigated the effects of the Trx-1 inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) on human acute myeloid leukemia cells (AML) and the sensitivity of cells to arsenic trioxide (As2O3, ATO). Treatment of cells with a different concentration of PX-12 for 48 h resulted in growth inhibition, the induction of apoptosis and increased the levels of activated caspase-3 expression in AML cell lines HL-60, NB4, U937 and primary AML cells in a dose-dependent manner. In addition, PX-12 enhanced the sensitivity of U937 cells to ATO. These results suggest the effects of Trx-1 inhibitor PX-12 to induce apoptosis in AML cells and therapeutic potential in AML by enhancing the sensitivity of cells to ATO.     

白血病细胞纤溶活性及机制的研究

目的： 研究白血病细胞纤溶活性及其与尿激酶受体(uPAR)和膜联蛋白Ⅱ(annexinⅡ)表达的关系。方法: 用发色底物法测定NB4、SHI-1、K562、Jurkat、Raji细胞与纤溶酶原相互作用后白血病细胞的纤溶活性。用流式细胞术检测NB4、SHI-1、K562、Jurkat、Raji细胞表面uPAR和annexinⅡ的表达。以RT-PCR方法检测uPAR基因和annexinⅡ基因在NB4、SHI-1、K562、Jurkat、Raji细胞系中的表达。结果: SHI-1细胞和NB4细胞的纤溶活性明显高于Raji、K562和Jurkat细胞的纤溶活性。uPAR和annexinⅡ在NB4细胞的表达率分别为(13.15±1.61)%与(95.97±1.19)%，在SHI-1细胞的表达分别为(99.00±0.26)%与(90.35±2.15)%，在K562、Jurkat、Raji细胞的表达均低。NB4细胞annexinⅡ的基因表达较SHI-1细胞高，K562和Jurkat细胞中未检测到annexinⅡ的基因表达。NB4和SHI-1细胞uPAR基因表达高于Jurkat和K562细胞，Raji细胞中未检测到uPAR基因的表达。结论: NB4与SHI-1白血病细胞株存在高纤溶活性，与annexinⅡ和uPAR的表达增高相关，这可能是早幼粒细胞白血病与急性单核细胞白血病患者在临床上易发生出血及并发弥散性血管内凝血的一个重要原因。