华蟾素诱导NB4细胞凋亡及其作用机制

目的研究华蟾素(cinobufagin)对人早幼粒白血病细胞株NB4的诱导凋亡作用,并探讨可能的分子机制.方法通过细胞计数观察华蟾素对细胞的生长抑制作用,荧光染色观察细胞凋亡的形态学改变,DNA凝胶电泳观察DNA的片段化改变,流式细胞术定量分析细胞凋亡.免疫荧光结合流式细胞术检测bcl-2表达、半定量RT-PCR检测Fas和FasL的表达.结果与对照组相比,1:100～1:25浓度的华蟾素能明显抑制NB4细胞生长.经华蟾素作用后,NB4细胞出现凋亡典型的形态学改变,核浓聚、核碎裂、凋亡小体形成;DNA电泳出现凋亡特有的"梯子"条带;流式细胞术也证实凋亡的存在.在经1:100、1:50、1:25浓度的华蟾素作用2 d后,细胞凋亡率分别为12.49%、18.53%、37.76%.凋亡细胞的bcl-2的表达下调,Fas表达上调,FasL表达无变化.结论华蟾素能抑制NB4生长并诱导凋亡,可能是通过下调bcl-2的表达,增加Fas的表达来实现.     

三磷酸腺苷诱导U937细胞凋亡机制的探讨

目的：探讨三磷酸腺苷(ATP)诱导U937细胞凋亡过程中p73 mRNA的表达变化，以进一步研究p73基因在U937细胞凋亡中的作用。方法：用ATP诱导U937细胞凋亡，凋亡指标采用细胞形态学、DNA片段电泳、流式细胞术检测DNA含量及细胞周期的变化等方法；采用半定量反转录PCR(RT-PCR)检测p73 mRNA的表达变化。结果：ATP可诱导U937细胞凋亡。0.25 g／L的ATP作用于U937细胞24 h，光镜下见细胞出现较明显的聚集现象，胞膜皱缩，细胞体积缩小，Wright's+Giemsa．染色见胞核浓缩、核碎裂等现象，DNA片断电泳见清晰的梯形条带；流式细胞仪检测：0.25 g／L ATP作用于U937细胞24 h、36 h、48 h，细胞的凋亡率分别为2.33％、11.90％、35.49％，并使细胞阻滞在G2～M+S期，而对照组细胞凋亡率仅为1.09％；半定量RT-PCR结果：与对照组相比，仅48 h组p73 mRNA的表达下调。结论：ATP可诱导U937细胞凋亡。在U937细胞凋亡早期，p73 mRNA表达差异无显著性。     

抗氧化剂阻抑肿瘤坏死因子诱导的U937细胞凋亡

目的：探讨抗氧化剂对肿瘤坏死因子(TNF)诱导的U937细胞凋亡的作用。方法：首先用TNF处理U937细胞，提取小片段DNA进行DNA断裂分析；继而以不同抗氧化剂和TNF与U937细胞共同孵育，用台盼蓝拒染法得到细胞存活曲线，并通过RT-PCR、Western blotting检测细胞的bcl-2表达。结果：抗氧化剂可抑制TNF诱导的U937细胞凋亡，上调bcl-2的表达．结论：活性氧中间产物是TNF诱导细胞凋亡的中介体之一。     

丁酸钠增强U937细胞凋亡敏感性的分子机制研究

目的 探讨丁酸钠（NaBu)对细胞周期检测点效应及对U937细胞凋亡的敏感性。方法 以U937-ASPI3K（ATM阴性）,U937-pZeosv2( )（野生型ATM基因）两种U937的变异细胞系作为细胞模型。用免疫沉淀及激酶活性测定p38MAPK,ERK1的激酶活性。用免疫印迹分析Bad磷酸化灭活。结果 经NaBu预处理的U937-pZeosv2( )细胞经^137Gs照射后。细胞凋亡敏感性呈NaBu剂量依赖性增强,这种增强效应可被p38MAPK阻滞剂OLM阻断,但不能被p34cdc2激酶的特异性抑制剂ALP及CDK2阻滞剂CDK-2-Ⅰ阻断,经NaBu预处理的U937-ASPI3K细胞经^137Cs照射后,细胞凋亡敏感性进一步增强,这种增强效应可被OLM阻断,放射线可显著增强p38MAPK激酶活性,抑制ERK1激酶活性;NaBu预处理与放射线联用后,对p38MAPK激酶活性的增强有极其显著的协同效应,放射线诱导U937-ASPI3K细胞Bad蛋白磷酸化灭活,在NaBu协同作用下,Bad蛋白磷酸化灭活效应进一步增强。结论 NaBu通过p38MAPK激酶活性,增强细胞凋亡敏感性,该效应与ATM基因是否缺失无关,与ATM失活增强的细胞凋亡敏感性各为独立通路。     

右旋柠檬烯诱导人白血病细胞凋亡作用机制的初步研究

目的:探讨右旋柠檬烯(d-limonene)诱导人白血病细胞凋亡的作用机制.方法:以人白血病细胞株K562和HL-60为研究对象,采用MTT法及锥虫蓝染色法检测d-limonene对细胞增殖的影响,Annexin V/PI双重染色法检测其对细胞凋亡的影响,Western印迹法检测线粒体凋亡途径相关蛋白的表达.结果:d-Limonene作用后K562、HL-60细胞凋亡率呈时间及剂量相关性增加;且处理组Bcl-2/Bax比值下降,细胞质中细胞色素c表达增高,caspase-9表达以及caspase-3分裂明显增高.结论:d-limonene能够诱导人白血病细胞凋亡,线粒体凋亡途径的激活可能是其凋亡诱导的主要机制.     

罗格列酮对U937细胞生长抑制作用及其机制的探讨

目的:探讨过氧化物酶体增殖因子活化受体γ(PPARγ)配体罗格列酮(ROS)对人急性单核细胞白血病U937细胞生长抑制作用及其作用机制.方法:体外培养人急性单核细胞白血病U937细胞,应用不同浓度的罗格列酮处理人急性单核细胞白血病U937细胞,采用MTT比色法测定药物对细胞增殖的抑制作用;用流式细胞术、AnnexinV/PI双染色法现察细胞凋亡率;并分析细胞周期改变;RT-PCR法分析PPARγ/mRNA表达.结果:ROS浓度>80#mol/L时,对U937细胞产生明显的增殖抑制作用,P<0.05;AnrlexinV/PI双染色法观察细胞凋亡率>50%,并将细胞周期阻滞在G1期,P<0.05;RT-PCR检测U937细胞中PPAR7 mRNA表达无明显变化.结论:ROS浓度>80 μmol/L时,对U937细胞产生明显的增殖抑制作用及诱导凋亡作用,同时将细胞周期阻滞在G1期,但是PPARγ mRNA表达无明显变化,推测可能与激活PPARγ表迭无明显相关,可能存在另外的信号转导途径.     

转染外源性Fas配体基因的正常骨髓CD34+细胞诱导U937细胞凋亡的实验研究

目的:观察转染外源性FasL的CD34 细胞对白血病细胞系U937的凋亡诱导作用,探讨通过向CD34 细胞转染FasL以增强移植后GVL效应的可能性.方法:免疫磁珠法分选骨髓CD34 细胞并进行体外扩增培养;FasL基因逆转录病毒途径转染CD34 细胞,并与白血病细胞U937混合培养,加入或不加入柔红霉素、阿糖胞苷,借助TUNEL、流式细胞仪检测细胞的凋亡率.结果:逆转录病毒上清转染后48～72h,流式细胞仪检测24.2%±2.4?34 细胞表达FasL(P＜0.01)(未转染的CD34 细胞FasL阳性率为7.6%±1.1%).未转染或转染外源FasL的CD34 细胞与白血病细胞U937以1:1的比例混合培养18h后,细胞凋亡率分别为5.0%±1.3%、10.8%±0.6%(P＜0.01);FasL CD34 DNR或FasL CD34 Arac作用后,细胞凋亡率分别为13.4%±1.0%(P＜0.05)、17.95%±1.3%(P＜0.01).结论:表达外源性FasL的骨髓CD34 细胞可诱导白血病细胞U937凋亡.提示植入转染外源性FasL基因的骨髓CD34 细胞可增强BMT后的GVL效应.     

油茶皂苷体外诱导人白血病Jurkat细胞凋亡及其可能机制

目的:探讨油茶皂苷在体外诱导人白血病Jurkat细胞的凋亡及其可能机制。方法:将不同浓度的油茶皂苷作用于人白血病Jurkat细胞,应用细胞计数法检测其对细胞增殖的影响,FCM检测细胞周期分布和细胞凋亡率,蛋白质印迹法检测细胞caspase-3、多聚ADP-核糖聚合酶[poly(ADP-ribose)polymerase,PARP]、p-Bcl-2、Bcl-2、Bax和caspase-9蛋白的表达。结果:1～16μg/mL油茶皂苷可抑制Jurkat细胞的增殖,呈剂量依赖性。1～4μg/mL油茶皂苷作用Jurkat细胞24h后,G0/G1期和G2/M期细胞比率下降,S期细胞比率上升,细胞凋亡率随着油茶皂苷浓度的增加而上升;Jurkat细胞中,p-Bcl-2和Bcl-2蛋白的表达水平下调,caspase-3、PARP和caspase-9蛋白的表达水平上调,Bax蛋白的表达水平无明显变化。结论:油茶皂苷能抑制人白血病Jurkat细胞增殖和诱导其凋亡,其作用机制可能与细胞凋亡的线粒体途径有关。     

抗氧化剂阻抑肿瘤坏死因子诱导的U937细胞凋亡

近几年来,细胞凋亡（ａｐｏｐｔｏｓｉｓ）的研究日益深入,凋亡的调控可能与细胞类型及诱发因素有关,某些因素具有普遍性。研究表明,活性氧中间体（ｒｅａｃ－ｔｉｖｅｏｘｙｇｅｎｉｎｔｅｒｍｅｄｉａｔｅｓ,ＲＯＩ）是细胞凋亡的媒介之一,许多理化因素诱导的细胞...     

靶向HOXA9基因shRNA慢病毒载体对U937细胞增殖与凋亡影响观察

目的：观察慢病毒介导的短发夹RNA（shorthairpinRNA,shRNA）沉默同源盒A9（homeoboxA9,HOXA9）基因对U937细胞增殖和凋亡的影响。方法：前期从4条针对HOXA9基因的RNA干扰（RNAinterference,RNAi）慢病毒载体中筛选出了l条敲减效率最高的包装成慢病毒H0xA9／GVll8RNAi—LV#2。实验分为对照组（control,CON）、阴性对照组（negativecontrol,NC）及敲减组（knockdown,KD）。HOXA9／GVll8RNAi-LV#2感染U937细胞5d后,FCM检测U937细胞感染效率,应用实时荧光定量PCR法检测HOXA9mRNA的沉默效率,蛋白质印迹法检测HOXA9蛋白的表达;MTT法和绘制细胞生长曲线检测细胞增殖抑制情况;FCM检测细胞凋亡及细胞周期;RT-PCR法检测细胞c-mycmRNA及p27mRNA表达情况。结果：慢病毒感染效率〉90％,KD组细胞中HOXA9mRNA的沉默效率〉55％,HOXA9蛋白的表达量明显减少,其中KD组相对表达水平达（0．223±0．024）,显著低于NC组（0．884±0．009）及CON组（0．891±0．013）,差异有统计学意义,F=1566．202,P〈0．001。MTT结果显示,KD组细胞的IR值为（40．469±1．756）％,明显高于NC组及CON组（F=1311．928,P〈0．001）,细胞生长曲线表明该组细胞生长速度减慢;FCM结果显示,KD组、NC组及CON组凋亡率分别为（26．762±2．245）％、（6．819±0．547）％和（6．113±0．349）％,KD组与NC组（q=25．501,P〈0．001）及CON组（q=26．418,P〈0．001）比较差异均有统计学意义,提示该载体显著增加细胞凋亡率;其细胞周期G0／G1期细胞比例较CON组及NC组明显增加,提示该载体使细胞周期阻滞于G0／G1期,F=27．769,P=0．001;RT-PCR结果表明,U937细胞KD组较CON组及NC组c-mycmRNA表达水平下降,p27mR—NA表达水平上升。结论：靶向HOXA9基因shRNA慢病毒载体能稳定沉默HOXA9表达,可显著抑制U937细胞增殖并促进其凋亡,同时可下调c-myc表达,上调p27表达,使细胞周期阻滞于G0／G1期。     

Actinonin induces apoptosis in U937 leukemia cells

S100A4 has multiple functions in cell cycle progression and cell motility, and has been implicated in cancer invasion. In this study, we examined the expression of S100A4, E-cadherin and its related proteins in oral squamous cell carcinoma (SCC) cell lines with different invasive phenotypes, grade 4C and 4D. Furthermore, grade 4C OSC-19 cells expressing E-cadherin were transfected with S100A4-expression vector and the expression of E-cadherin-related proteins in the stable clone was examined to elucidate the relationship between S100A4 and E-cadherin. Constitutive over-expression of S100A4 in stable transformant of OSC-19 (OSC-19/S100A4) cells led to down-regulation of E-cadherin and β-catenin. Furthermore, grade 4D invasive cell lines (HOC313 and TSU) expressing S100A4 mRNA did not express E-cadherin, P-cadherin, and β-catenin, while γ-catenin protein was only weakly expressed. Thus, the mRNA expression of E-cadherin was reversely correlated with S100A4 expression in oral SCCs. Interestingly, vascular endothelial growth factor-C was up-regulated in OSC-19/S100A4 cells. In summary, S100A4-mediated regulation of E-cadherin expression may play an important mechanism in invasion and metastasis of oral SCC.     

Molecular Mechanism of Differentiation and Apoptosis of U937 Cells Induced by 12-O-Tetradecanoylphorbol-13-Acetate

OBJECTIVE To explore the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on differentiation, apoptosis and related molecular mechanisms in U937 myelomonocytic leukemia cells.METHODS Morphological changes were analyzed by phase contrast and light microscopy, expression of the monocytic differentiation maker CD11b by direct immunofluorescence staining, cell cycle distribution and apoptosis by flow cytometry, and expression of bcl-2, Bax, survivin and p21Cip1/Waf1proteins by Western analysis.RESULTS Treatment of U937 cells with 10 nmol/L TPA induced cell adherence. The adherent cells showed G0/G1 cell cycle arrest (69.0% at 24h vs 52.1% control; P< 0.01),and morphologic changes and increased expression of the monocytic differentiation marker CD11b (63.0% at 72 h vs15.3% control; P< 0.01 ). In addition to these effects, about 20% of the cells still remained in suspension and exhibited a time-dependent increasing apoptosis, which reached 70.3% after 72 h of treatment ( P< 0.01 ). TPA treatment for 24 h induced expression of p21Cip1/Waf1 in the adherent cells, but not in the non-adherent cells. Furthermore, bcl-2 and survivin expression declined in 24 h-TPA-treated non-adherent cells compared with untreated control and adherent cells, whereas no change in the expression of Bax was detected.CONCLUSION TPA induces both differentiation and apoptosis in U937 cells,which may be related to the upregulation of p21Cip1/Waf1 and downregulation of bcl-2 and survivin expression.     

肿瘤坏死因子α致肝细胞凋亡效应及其机制的研究

Objective: To investigate the apoptosis effect of TNF-a on HepG2 cells and its mechanism in vitro.Methods: HepG2 cells were treated with TNF-α(400 U/mL).HepG2 cells treated with TNF-αfor 24 h and apoptosis was proved by DNA fragments on gel electrophoresis.Fluorescent quantitative real-time PCR was used to detect Fas and FasL expression.HepG2 cells treated by TNF-αwere co-cultivated with normal HepG2 cells.Apoptosis of HepG2 cells was determined by the method of FACS.Results: After 24 h TNF-αtreatment, DNA was collapsed into fragments to form DNA ladder in gel electrophoresis; FasL expression increases and induces HepG2 cells apoptosis.By FACS, 98.4% of the co-cultivated cells were apoptosis, but 16.5% cells in control group were apoptosis.Conclusion: TNF-αcan induce apoptosis of HepG2 cells in vitro.FasL expression on TNF-αpre-treated HepG2 cells increased and these cells can lead normal HepG2 cells to apoptosis.This may attribute to the cure of virus hepatitis and hepatoma.     

维甲酸提高髓母细胞瘤的化疗敏感性及其分子机制

目的　观察经全反式维甲酸 (RA)诱导分化的髓母细胞瘤细胞系Med 3对顺铂 (DDP)的化疗敏感性 ,死亡性质及其内在的分子机制。方法　分析RA、DDP和两者配伍对Med 3细胞形态和增殖的影响 ,观察被处理细胞的死亡特点并检测各处理因素对凋亡相关基因Fas和Fas配体 (FasL)表达水平和表达方式的影响。结果　可溶型和膜型FasL在正常培养和被处理的Med 3细胞中均呈阳性 ,Med 3细胞产生可溶型Fas并呈胞浆阳染。与DDP不同 ,RA可明显上调Fas表达并使Fas蛋白从胞浆移至细胞表面 ;但仅在它与DDP联用时 ,才能诱导靶细胞凋亡。结论　分化诱导剂RA通过修饰Fas基因表达形式 ,提高髓母细胞瘤Med 3细胞对DDP的凋亡敏感性。RA/DDP配伍方案可能成为临床治疗髓母细胞瘤的新途径。     

Tetraarsenic oxide induces apoptosis in U937 leukemic cells through a reactive oxygen species-dependent pathway

In the present study, we investigated the effect of tetraarsenic oxide (As4O6, 2,4,6,8,9,10-Hexaoxa-1,3,5,7-tetraarsatricyclo[3.3.1.13,7]decane) upon induction of apoptosis in arsenic trioxide (diarsenic oxide, As2O3) resistant U937 leukemic cells. As4O6 induced apoptosis in U937 leukemic cells at much lower concentrations than As2O3 via an early increase of cellular reactive oxygen species (ROS), and a decrease in cellular mitochondrial membrane potential, followed by cytochrome c release and caspase-3 activation. As4O6 generated ROS and induced caspase-3 activation more potently than As2O3 in U937 cells. Incubation of the cells with N-acetyl-L-cysteine and catalase resulted in significant suppression of As4O6-induced apoptotic cell death. These results show that the generation of ROS leads to the consequences associated with apoptosis induced by As4O6. In conclusion, As4O6 might be a new arsenic compound which may induce apoptosis in U937 leukemic cells by activating unique apoptotic signaling mediated by ROS more potently than As2O3, and deserves further evaluation.     

二甲双胍体外诱导人肝癌Huh-7细胞凋亡及其作用机制

目的 探讨二甲双胍对人肝癌Huh-7细胞增殖、凋亡的影响及其可能的作用机制.方法 二甲双胍干预Huh-7细胞后,四甲基偶氮唑蓝(MTT)法检测其对细胞活力及生长抑制的影响;Western blot检测凋亡相关蛋白及CD133蛋白的变化;流式细胞术检测细胞凋亡及肝癌干细胞相关标志物CD133的表达;无血清悬浮培养观察二甲双胍对肿瘤干细胞球形成的影响;逆转录聚合酶链反应(RT-PCR)检测其对肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达的影响.以加入与实验组相同的培养液但未给予药物处理的组为对照组.结果 MTT检测结果显示,与对照组比较,随着二甲双胍浓度的增高,肝癌Huh-7细胞的增殖率下降,差异均有统计学意义(均P＜0.05).10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的早期凋亡率为(22.29±0.80)％,对照组的早期凋亡率为(6.70±0.50)％,差异有统计学意义(P ＜0.05);10 mmol/L二甲双胍干预48 h后肝癌Huh-7细胞的晚期凋亡率为(13.87±1.20)％,对照组的晚期凋亡率为(1.10±0.02)％,差异有统计学意义(P＜0.05).25 mmol/L二甲双胍干预48 h后细胞的早期凋亡率为(15.28±2.10)％,晚期凋亡率为(25.89±2.30)％,均高于对照组(均P＜0.05).Western blot检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h后,p-Akt、Bcl-2/Bax比值表达下调,PTEN表达上调.随着二甲双胍浓度的增加,CD133蛋白的表达下调.流式细胞仪检测结果显示,25 mmol/L二甲双胍干预Huh-7细胞48 h前后,Huh-7细胞中CD133+细胞的表达率分别为(40.7±2.1)％和(14.6±1.85)％ (P ＜0.05).在低黏附培养皿、无血清培养液的生长环境中,对照组细胞球体积较大,中心厚重,周围清亮,折光度强;10mmol/L二甲双胍组细胞球体积较小,球体折光度较弱,细胞球数量少于对照组.RT-PCR检测结果显示,肿瘤干细胞相关基因CD133、β-catenin、ABCG2 mRNA表达水平均下调.结论 二甲双胍能显著抑制人肝癌Huh-7细胞增殖,促进凋亡.其机制可能与抑制肝癌PI3 K/Akt信号通路和CD133+肿瘤干细胞的表达有关.     

Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells

PURPOSE: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. METHODS AND MATERIALS: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (DeltaPsi) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). RESULTS: Time courses of the apoptotic rates, caspase activation, and DeltaPsi indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. CONCLUSIONS: Irradiation of U937 cells at different X-ray doses induced two different types of apoptotic cell death, premitotic apoptosis and postmitotic apoptosis, which are characterized by the time course and cell-cycle specificity. Decision concerning these two types of apoptotic cell death may be made by the difference in the magnitude of cell damage following X-irradiation.     

Bcl-2 overexpression attenuates SP600125-induced apoptosis in human leukemia U937 cells

SP600125 is a specific inhibitor of c-Jun N-terminal kinase (JNK) that is known to strongly induce apoptosis and block cell cycle progression in G₂/M phase. In this study, we demonstrated that treatment of U937 cells with SP600125 resulted in significant G₂/M cell cycle arrest that was due to decreased cyclin B1 and cdc25c protein levels. Moreover, SP600125 promoted LDH release and DNA fragmentation that was associated with caspase-3 activation and degradation of its substrates. In contrast, overexpression of the antiapoptotic protein Bcl-2 rendered leukemia cells resistant to SP600125-induced apoptosis, but more sensitive to G₂/M phase arrest and endoreduplication (>4N DNA). Overexpression of Bcl-2 significantly inhibited SP600125-induced caspase-3 activation and degradation of its substrates, and sustained expression levels of the IAP-2 proteins following SP600125 treatment. The inhibitory effect of Bcl-2 on apoptosis was attenuated by treatment with the small molecule Bcl-2 inhibitor, HA14-1. These data provide important mechanistic insights related to Bcl-2-mediated resistance to SP600125-induced apoptosis, and induction of G₂/M phase arrest and endoreduplication.