亚硒酸钠诱导NB4细胞凋亡及分化

急性早幼粒细胞白血病（APL）约占成人急性髓性白血病（AML）的10％,APL的特征为在粒系发育过程中,细胞的分化被阻滞于早幼粒阶段。有研究发现：与其他白血病细胞株相比,NB4细胞（APL的细胞株）抗氧化应激的能力较弱,一些临床上有效治疗APL的药物如AS2O3,可通过诱导氧化应激进而诱导NB4细胞凋亡。已经发现,作为有效的癌化学预防剂,一些硒化合物在体外能抑制多种肿瘤细胞的生长,而含硒酶或蛋白在细胞的氧化调节过程中扮演着重要的角色。本研究以NB4细胞为模型,研究了单独应用Na2SeO3在2、5、10和20μmol／L等浓度下,或…     

靶向PML-RARα融合基因的siRNA对急性早幼粒细胞白血病细胞NB4活性的影响

通过靶向PML-RARα融合基因的小干扰RNA(siRNA)的体外干扰,研究其对急性早幼粒细胞白血病细胞NB4活性的影响。设计合成5对靶向PML-RARα融合基因的siRNA,转染NB4细胞,采用MTT比色法、软琼脂细胞集落形成实验检测siRNA对NB4细胞的增殖抑制作用,用流式细胞术测定细胞的凋亡,结果显示siRNA显著抑制了NB4细胞的增殖并有效诱导细胞凋亡。靶向PML-RARα融合基因的siRNA有望成为治疗急性早幼粒细胞白血病的有效方法。     

SeO2诱导白血病细胞凋亡过程中对细胞内活性氧和Ca2+水平影响

目的：研究SeO2对急性早幼粒细胞白血病细胞株NB4、红白血病细胞K562、急性粒细胞白血病细胞株HL-60的增生、凋亡、活性氧(ROS)及Ca2+水平等的影响。 方法： 采用不同浓度SeO2（3-30 μmol/L）分别处理3种白血病细胞，用流式细胞术测定细胞凋亡率、细胞中ROS和Ca2+的水平。 结果： 10和30 μmol/L SeO2能抑制3种细胞增生，30 μmol/L SeO2作用48 h能使54.0%的NB4细胞、46.5%的K562细胞和49.6%的HL-60细胞发生凋亡。同时下调细胞内ROS和Ca2+水平。NB4和HL-60细胞中ROS阳性细胞比例随SeO2浓度增加而减少，K562中只有30 μmol/L SeO2才能使ROS出现明显下降。10、30 μmol/L SeO2能使NB4和HL-60细胞内Ca2+水平逐步下降。K562中只有30 μmol/L SeO2才能使细胞内Ca2+水平出现明显下降。 结论： SeO2对3种白血病细胞均有诱导凋亡作用，在凋亡过程中涉及细胞内ROS及Ca2+水平下降。     

PPARγ激动剂对白血病NB4细胞的诱导凋亡作用及其作用机制

目的探讨过氧化物酶体增殖物激活受体γ（PPARγ）激动剂罗格列酮（rosiglitazone,RGZ）对白血病NB4细胞的诱导凋亡作用及其作用机制。方法以不同浓度的RGZ作用于体外培养的NB4细胞0、24、48及72h．应用MTF法检测细胞生长抑制率,用Annexin V／PI双染法检测细胞凋亡,并对细胞凋亡前后P53蛋白的表达水平进行检测。结果RGZ可显著抑制细胞的生长及诱导细胞发生凋亡,呈现出明显的量-效与时-效关系。在细胞凋亡的同时,P53蛋白的表达水平明显升高。结论PPARγ激动剂RGZ能显著抑制NB4细胞的生长并诱导细胞发生凋亡,升高促凋亡蛋白P53的表达水平可能是RGZ诱导NB4细胞发生凋亡的重要作用机制之一。     

白血病细胞中端粒酶抑制因子Pinx1的表达与端粒酶活性关系的研究

目的：研究端粒酶抑制因子Pinx1在急性白血病细胞中的表达和在急性早幼粒白血病细胞株细胞NB4分化过程中的表达改变，分析其表达与端粒酶逆转录酶hTERT表达的关系，以了解白血病细胞中Pinx1对端粒酶的作用及可能机制。方法:荧光定量RT-PCR检测30例急性白血病细胞中Pinx1与hTERT mRNA的表达，进一步在ATRA诱导的急性早幼粒白血病细胞株细胞NB4分化过程中检测Pinx1及hTERT mRNA表达，分析两者之间的相关性。结果:Pinx1在急性白血病细胞中的表达（0.00312，5.42×10-4-0.024）明显高于正常人骨髓单个核细胞（7.89×10-4，0-0.00863，P＜0.01），与hTERT的表达呈正相关（r=0.296,P＜0.05）。Pinx1表达随NB4细胞分化逐渐下降，与hTERT呈正相关（r=0.900,P＜0.05） 。结论：Pinx1虽然为端粒酶活性的抑制因子，但在白血病细胞中与端粒酶活性的调控方向一致，提示Pinx1的表达改变可能是继发于hTERT的一种负反馈反应，目的是保持端粒酶活性的稳定，具体机制尚待进一步研究。     

对比硫化砷对两种白血病细胞株凋亡和hTERT-mRNA表达的影响

目的:对比硫化砷对慢性粒细胞白血病细胞株K562和急性早幼粒细胞白血病细胞株NB4两种细胞的作用是否存在差异及其机制.方法:PCR-ELISA法测定端粒酶活性;半定量RT-PCR检测hTERT mRNA的表达;流式细胞术测量细胞周期、凋亡.结果:0.15～0.6 mg/L硫化砷(72 h)可在诱导NB4细胞凋亡的同时,下调细胞端粒酶活性和hTERT-mRNA表达,同样作用对于K562细胞需要硫化砷浓度达到0.3～3 mg/L.0.3 mg/L硫化砷(72 h)可引起NB4细胞出现G2/M期细胞比例增高.而K562细胞需要在1.5mg/L硫化砷诱导下出现G2/M期细胞比例增高.结论:端粒酶系统可能是硫化砷诱导NB4和K562细胞凋亡的途径之一;由硫化砷诱导的G2/M期阻滞可能与端粒酶活性的显著下降相关.NB4和K562细胞对硫化砷的敏感性存在明显差异,具体机制有待进一步研究.     

Bcl-2基因反义核酸增强三氧化二砷对NB4细胞凋亡效应

目的:研究和探索bcl-2基因反义核酸(bcl-2ASODN)是否能提高三氧化二砷(As₂O₃)对NB4白血病细胞的凋亡效应。方法:采用微量细胞培养的方法,观察反义核酸、砷剂单独以及反义核酸、砷剂联合培养NB4细胞的生物效应和凋亡形态变化;用免疫组化的方法,结合流式细胞技术,测定NB4细胞的DNA和Bcl-2蛋白的变化。结果:As₂O₃(0.25μmol/L)+bcl-2ASODN(10.0μmol/L)联合诱导NB4细胞凋亡作用显著强于单用As₂O₃(0.25μmol/L)或bcl-2ASODN(10.0μmol/L)(P＜0.01)。流式细胞仪检测显示,联合用药Bcl-2蛋白表达亦明显少于反义核酸、砷剂单独给药组(P＜0.01)。结论:bcl-2基因反义核酸能明显提高和显著增强As₂O₃对NB4白血病细胞的致凋亡效应。     

我国人类白血病研究获重大成果

我国人类白血病研究获重大成果由陈竺、陈赛娟两位教授领衔研究的“人类白血病诱导分化和凋亡的细胞及分子机制研究”实现重大突破：不仅发现了三氧化二砷（俗称砒霜）可以治疗初发的急性早幼粒白血病患者，而且对已经产生维甲酸耐药或用维甲酸缓解后复发的患者，仍可再次...     

蝌蚪提取液诱导HL-60细胞凋亡及相关基因的表达

以往实验证明蝌蚪提取液 (T871)具有抑制肿瘤细胞生长并诱导其分化的作用[1 ] 。为进一步探讨蝌蚪提取液的抗肿瘤作用 ,本实验观察了T871诱导人早幼粒白血病细胞系(HL 6 0 )细胞凋亡作用及其凋亡过程中相关癌基因的变化。1　材料和方法1 1　人早幼粒白血病细胞系 (HL 6 0 )细胞的培养 :用含 10 %胎牛血清的RPMI 16 4 0培养基培养 (含青霉素 10 5U L ,链霉素10 5U L ,L 谷氨酰胺 2mol L)。临用前用 8%NaHCO3调pH至7 0～ 7 2 ,37 0℃ ,饱和湿度 ,5 %CO2 条件下悬浮培养。1 2　蝌蚪提取液 (T871)的制备 :取池塘黑色有尾无腿活蝌蚪(经…     

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。     

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).     

Spontaneous migration of acute promyelocytic leukemia cells beneath cultured bone marrow adipocytes with matched expression of the major histocompatibility complex

Leptin, secreted by adipocytes in bone marrow stimulates proliferation of acute myeloid leukemia cells. PML-RARalpha-expressing acute promyelocytic leukemia (APL) cells express high levels of leptin receptor (OB-R). Adipocytes derived from mesenchymal stem cells (MSC) protect APL cells from drug-induced apoptosis partially through the interaction of the membrane-bound form of leptin and OB-R. We report that when NB4 APL cells migrated beneath a cultured MSC-derived adipocyte layer, apoptosis in most of the cells below the adipocytes was blocked. Major histocompatibility complex (MHC) expression of NB4 cells and MSCs matched (both were HLA-DR negative and HLA-ABC positive). This match in MHC expression may play a role in the intimate cell-to-cell interactions that support the anti-apoptotic effects of the membrane-bound leptin signaling pathway.     

亚硒酸钠诱导的人白血病耐药细胞株MR2的生长抑制及凋亡

本文以人急性髓系白血病M3型耐药细胞株（抗全反式维甲酸ATRA）MR2为研究对象，通过细胞生长增殖分析，细胞凋亡相关指标的检测，细胞内活性氧自由基含量的测定等，探讨亚硒酸钠对MR2细胞的凋亡诱导作用及其作用机制。结果发现：（1）亚硒酸钠浓度大于10μmol/L时对细胞株有明显的生长抑制作用，大于20μmol/L时有明显的凋亡作用，并呈剂量时间依赖性增加。40μmol/L的亚硒酸钠作用细胞60h后，可使凋亡百分率超过80％；（2）亚硒酸钠作用细胞一定时间后可使胞内自由基水平增加，并在24h时出现最大值，以上结果表明亚硒酸钠对MR2细胞的生长抑制及凋亡诱导作用可能是通过氧化应激实现的。     

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.     

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.     

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.     

三氧化二砷对急性早幼粒细胞白血病细胞分泌细胞因子的影响与意义

为了探讨细胞因子在三氧化二砷诱发APL细胞白细胞升高的作用 ,首先按照FAB细胞形态学和细胞遗传学标准选择APL患者。常规Ficoll密度梯度法分离骨髓和PBMC ,体外培养 1h去除贴壁细胞。体外IL 1β、IL 6、IL 8、TNF α和G CSF分泌水平采用ELISA方法进行测定 ,NBT还原实验用以检测APL细胞分化状态。直接细胞计数法和MTT法观察细胞增殖变化。研究结果证实体外 10 6M或体内以 10mg/d三氧化二砷治疗 96h后 ,APL细胞分泌IL 1β和G CSF平均水平升高 (P <0 0 5 ) ,IL 6和IL 8水平降低 (P <0 0 5 ) ,TNF α水平无明显变化 (P >0 0 5 )。进一步分析结果表明 ,体外APL细胞的增值比率与IL 1β或G CSF分泌升高比率呈现正相关 ,检测到IL 1β或G CSF患者的细胞数量增加比率高于未检测到IL 1β或G CSF组 ,说明IL 1β或G CSF分泌增加在三氧化二砷诱导后的APL细胞增殖中起着一定作用     

乌苯美司下调c-myc表达与其增强全反式维甲酸诱导NB4细胞

目的： 了解氨肽酶抑制剂乌苯美司（bestatin）能否增强全反式维甲酸（ATRA）对NB4细胞的诱导分化作用，及此过程中NB4细胞c-myc mRNA表达水平的改变。 方法： MTT法检测药物抑制细胞生长的作用。流式细胞仪测细胞表面分化抗原CD11b及四氮唑蓝（NBT）还原实验检测NB4细胞的分化。RT-PCR检测细胞c-myc mRNA表达水平。 结果： 50 mg/L、75 mg/L、100 mg/L乌苯美司与10 nmoL/L ATRA联合处理72 h，均能明显增强NB4细胞的NBT还原能力，与10 nmoL/L ATRA组差异显著（P<0.05，P<0.01）。从48 h到96 h，100 mg/L乌苯美司时间依赖性地增强10 nmoL/L ATRA诱导NB4细胞的NBT还原能力，与相应时点10 nmoL/L ATRA组差异明显（P<0.01）。100 mg/L乌苯美司与10 nmoL/L ATRA联合应用72 h，NB4细胞CD11b表达率明显高于10 nmoL/L ATRA组（P<0.01）。50 mg/L、75 mg/L、100 mg/L乌苯美司与10 nmoL/L ATRA联合处理4 h，NB4细胞c-myc mRNA表达水平明显低于单用ATRA组（P<0.05，P<0.01）；药物联合应用各组NB4细胞的c-myc mRNA表达水平与NBT还原能力之间呈负相关（r=-0.917,P<0.05）。 结论： 乌苯美司可能通过下调NB4细胞c-myc mRNA的表达，从而增强ATRA诱导NB4细胞分化的作用。     

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.     

Inhibition on LS-174T cell growth and activity of telomerase in vitro and in vivo by arsenic trioxide.

Arsenic trioxide (As(2)O(3)) shows a significant therapeutic effect upon acute promyelocytic leukemia (APL) and can induce the apoptosis of NB(4) cells, which attracts scholars' great attention. Especially, the therapeutic effect on solid carcinoma has been paid more close attention to. The present study is to evaluate the effect of As(2)O(3) on human colorectal carcinoma cells (LS-174T cell) and the activity of telomerase in vitro and in vivo. This research made use of the electron microscope, polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), fluorescence-activated cell sorter (FACS), MTT in vitro and in vivo (LS-174T xenograft model of nude mice). With the increasing concentration of As(2)O(3), the ratio of living cells to dead cells decreased significantly, and the IC(50) value was 5.23mumol/L; cells of the experimental groups endured a series of morphological changes similar to the features of apoptosis. Apoptosis curve of FACS pictures appeared after 24h, and the cells showed apoptosis in a time-dependent manner; As(2)O(3) can inhibit the activity of telomerase of the cell extraction, obviously, in a concentration-dependent and time-dependent manner after 24h. As to the inhibition impact of As(2)O(3) on the xenograft model of nude mice in the two indexes, tumor volume and weight, there was a significant difference between As(2)O(3) and the control group; there was no difference between As(2)O(3) and the fluorouracil (5-FU) group; in the group of peritoneal injections of As(2)O(3), the cancer cells connected loosely with each other, nucleus changed markedly, and heterochromatin concentrated under the nucleus membrane. From the in vitro and in vivo experiment, we can see that As(2)O(3) inhibited LS-174T cell growth mainly by inducing cell apoptosis, partly by the inhibition of telomerase activity.