JWA基因在HL-60细胞定向分化中的表达及意义

为了研究JWA基因在HL-60细胞定向分化中的表达规律,探讨其在白血病细胞分化和凋亡中的意义及可能的作用机制,应用FCM检测HL-60细胞经ATRA（10^-6 mol/L）、Ara-C（10 ng/ml）、TPA（10^-8 mol/L）作用后2、4、6、8天CD13、CD14、CD15、CD11b改变及细胞周期变化;应用半定量RT-PCR及蛋白印迹技术检测JWA基因在HL-60细胞定向分化中的表达趋势以及相关凋亡因子Bcl-2、HSP27和HSP70的表达.结果表明：经上述药物分别处理HL-60细胞后,不同时段FCM相关指标检测结果证明其分别向粒、单核、巨噬细胞样分化.JWA基因表达呈时间依赖性升高;Bcl-2则呈时间依赖性下降;HSP70在ATRA及TPA组与JWA表达呈正相关,与Bcl-2呈负相关,而各组HSP27并无表达.ATRA处理HL-60细胞后JWA基因表达趋势与原代细胞相反,且不需要ATRA诱导分化作为前提.分别以Ara-C 10 ng/ml和Ara-C 20 ng/ml处理HL-60细胞后,JWA基因的表达呈反向变化,即前者呈时间依赖性增加,而后者则下降.结论：JWA基因在实现ATRA及TPA诱导白血病细胞分化和凋亡中可能具有双重调节作用;JWA基因表达的高低与Ara-C诱导分化作用及细胞毒作用有关;HL-60细胞与原代白血病细胞在诱导分化机制方面可能不尽相同.     

铁螯合剂诱导白血病细胞凋亡中caspase-3的变化

目的探讨铁螯合剂去铁胺（DFO）对诱导白血病细胞HL-60的分子机制。方法 2003年712月用钙黄绿素（calcein）检测HL-60细胞LIP。台盼蓝活细胞拒染实验进行活细胞计数及细胞存活率测定;光镜形态学观察及流式细胞仪（FCM）等方法检测HL-60细胞凋亡;比色法检测caspase-3（基于pNA标记底物的比色法）活性。结果①不同浓度的DFO作用于HL-60细胞后,随培养时间延长及DFO浓度的增加,动态铁池降低,细胞生存率逐渐下降,凋亡率增加,显示一定的时间剂量依赖性。②HL-60细胞在不同浓度的DFO作用下,caspase-3的活性逐渐升高。50、100μmol/LDFO作用于HL-60细胞24h,caspase-3酶活性升高明显,与对照组相比,有统计学意义（P〈0.001）;相关分析结果显示,HL-60细胞LIP的改变与caspase-3活性变化呈负相关系（r=-0.887,P〈0.05）。结论 DFO诱导白血病细胞凋亡的作用可能与螯合细胞内铁,降低细胞LIP,激活caspase-3,最终实施细胞凋亡密切相关。     

雷公藤红素下调HL-60细胞P-Akt与Cyclin D1蛋白表达及其诱导细胞凋亡的效应

本研究探讨雷公藤红素诱导HL-60细胞凋亡及其可能的作用机制。以不同浓度雷公藤红素(0.25-8.0μmol/L)分别作用于HL-60细胞24-72小时,采用MTT法检测细胞增殖活性;TUNEL荧光染色、流式细胞术观察雷公藤红素对HL-60细胞凋亡及周期的影响;Western blot、RT-PCR法分别检测雷公藤红素对HL-60细胞内Akt(P-Akt)及其下游分子Cyclin D1的蛋白、基因的表达水平。结果表明,雷公藤红素能明显抑制HL-60细胞增殖,具有浓度依赖和时间依赖性。此外,雷公藤红素以浓度依赖性方式诱导HL-60细胞凋亡,并伴随明显的凋亡细胞形态学改变。雷公藤红素的凋亡诱导可能与其诱导HL-60细胞周期阻滞于G0/G1期有关。雷公藤红素对P-Akt及CyclinD1蛋白及基因表达水平均有不同程度的抑制作用,该抑制作用呈明显的量效和时效关系。结论:雷公藤红素明显抑制HL-60细胞的增殖,并诱导其凋亡,其抗白血病效应可能与其下调P-Akt和Cyclin D1蛋白表达有关。     

塞来西布对HL-60细胞增殖、凋亡及表达VEGF影响的体外研究

目的研究塞来西布（Celecoxib）对急性髓系细胞白血病（AML）HL-60细胞的增殖抑制、诱导凋亡作用及其机制。方法以不同浓度的Celecoxib作用于体外培养的HL-60细胞,用MTT法检测细胞生长抑制率;用流式细胞仪分析凋亡细胞的百分率和细胞周期变化;QRT—PCR法检测VEGF表达水平。结果Celeeoxib以浓度依赖性方式有效地抑制HL-60细胞增殖,Celecoxib增加G0／G1期HL-60细胞百分率,降低S、G2期HL-60细胞百分率;Celecoxib能以浓度依赖性方式诱导HL-60细胞凋亡。当Celecoxib浓度大于40μmol／L时,随着Celecoxib浓度增高,HL-60细胞VEGF-mRNA的表达逐渐降低,呈浓度依赖性变化。结论Celecoxib对HL-60细胞具有显著的增殖抑制和诱导凋亡作用,增殖抑制可能在于阻断了细胞周期的进行,降低VEGF活性可能是其诱导凋亡重要作用机制之一。     

复方中药制剂对急性髓系白血病细胞诱导分化与增殖的作用

目的:研究复方中药制剂对白血病细胞的作用及其机制。方法:用复方中药制剂处理体外培养的髓系白血病细胞株,用CCK8的方法观察中药制剂对白血病细胞增殖的影响,瑞氏染色法观察细胞形态学的变化,流式细胞术检测细胞凋亡并测定细胞表面分化抗原CD11b的表达水平。结果:与单纯的4种白血病细胞系HL-60,MOLM-13,MV4-11,AML-M5相比较,不同中药浓度处理的这4种白血病细胞增殖能力减弱(P0.05)且其增殖抑制作用呈剂量依赖性(r=0.9236;r=0.7488;r=0.8889;r=0.8119);与单纯的HL-60和AML-M5白血病细胞系相比,药物处理的这2种白血病细胞有明显的分化形态改变;与单纯的HL-60白血病细胞系相比,IC50中药浓度处理的HL-60细胞表面抗原CD11B增加85%±7.13%;与单纯的AML-M5白血病细胞系相比,1.5μl和2μl剂量中药制剂处理的AML-M5细胞的凋亡率增加(P 0.05)。结论:复方中药制剂对白血病细胞具有抑制增值作用,其机制可能与诱导白血病细胞分化和凋亡作用相关。     

新型氨基甾体类对人粒系白血病HL-60细胞系的增殖抑制和分化诱导的作用

研究发现一种新型氨基甾体化合物,2β-(4’-甲基-1’-哌嗪基)-3α,17β二羟基-5α-雄甾烷(HY)可抑制HL-60细胞的增殖,并诱导该类细胞向巨噬样细胞分化。其主要证据如下:①细胞计数,集落计数及MTT检测显示抑制HL-60细胞的增殖;②液体培养6天后形态学显示诱导HL-60细胞向巨噬样细胞分化;③可诱导NBT反应阳性;④可诱导α-萘酚醋酸酯酶反应阳性;⑤流式细胞术显示可诱导CD11b和CD14的表达。结论提示该化合物具有治疗白血病的潜在价值。     

全反式维甲酸诱导白血病细胞分化对brd7基因表达的影响

为了探讨brd7基因与白血病细胞分化的关系及其在白血病细胞分化中的作用,本研究采用全反式维甲酸(ATRA)诱导HL-60和K562细胞系分化,通过Wright-Giema染色在显微镜下观察细胞形态学变化,应用流式细胞术分析细胞分化抗原CD11b的表达,以鉴定细胞分化程度,并在此基础上通过Western blot检测brd7基因在诱导分化前和细胞分化过程中蛋白表达水平的变化。结果发现,ATRA具有抑制HL-60细胞生长的作用,并可诱导HL-60细胞向粒系分化,在ATRA诱导细胞分化过程中HL-60细胞表面CD11b的表达水平逐渐上调,BRD7蛋白表达随着HL-60细胞的分化而增加;ATRA对K562细胞无诱导分化作用,brd7的表达也无明显变化。结论:随着HL-60细胞的分化,brd7基因表达上调,其机制有待进一步阐明。     

人IL-5诱导的STAT3酪氨酸磷酸化

目的分析人早幼粒细胞白血病细胞内STAT3的酪氨酸磷酸化活化情况。方法培养人早幼粒细胞白血病细胞株HL-60,分别用浓度为0,1.0,10,100 ng/ml的人白细胞介素（hIL）-5刺激,然后利用特异性抗体,用免疫沉淀法、聚丙烯酰胺凝胶（SDS PAGE）电泳及Western Blot方法进行检测。结果检测到HL-60细胞内不同浓度的STAT3表达。结论一定浓度的人IL-5能同时诱导HL-60细胞内STAT3α和STAT3α的酪氨酸（Y705）磷酸化,且在一定范围内这种诱导作用与IL-5的浓度呈正相关。     

二甲基亚砜诱导HL-60细胞分化的比较蛋白质组学研究

不同的诱导分化剂可诱导急性髓系白血病细胞系HL-60细胞向不同谱系的细胞分化[1].二甲基亚砜( DMSO)由于其溶解极性和非极性化合物能力极强而被广泛用作溶剂.目前,DMSO作为细胞低温保鲜剂(浓度一般在10％)可直接给患者注射[2].据最新报道,DMS0可通过活化转录因子促进成骨细胞分化[3].我们采用比较蛋白质组学方法,研究DMSO诱导HL-60细胞分化过程中蛋白质的差异表达,并探讨其可能的分子机制.     

丙戊酸诱导白血病HL-60细胞凋亡及其对h-tert基因表达的影响

为了探讨丙戊酸（valproic acid,VPA）对白血病HL-60细胞诱导凋亡的作用及其可能的机制,采用细胞毒性试验（CCK-8法）观察不同浓度VPA在不同作用时间对HL-60细胞增殖的影响,采用荧光显微镜检及流式细胞术检测细胞凋亡,并观察VPA作用后HL-60细胞端粒酶亚单位h-tert基因、凋亡相关蛋白表达和caspase-3活性的变化。结果表明：VPA呈剂量依赖性抑制HL-60细胞增殖（r=-0.87）.,诱导细胞凋亡;同时,抗凋亡蛋白BCL-2表达明显下降,促凋亡蛋白BAX表达上调,caspase-3活性增强,h-tert mRNA表达逐渐下降,HL-60细胞的凋亡率与h-tert mRNA表达呈负相关。结论：VPA可抑制白血病HL-60细胞增殖,诱导细胞凋亡;VPA可能通过下调h-tert mRNA、BCL-2蛋白表达,上调BAX表达及增强caspase-3活性而发挥抗白血病作用。     

环孢霉素A逆转人白血病耐药细胞系HL-60／ADM后氧自由基水平的变化

本研究旨在探讨将抗阿霉素的人急性早幼粒细胞白血病细胞系HL-60／ADM的耐药性逆转后细胞内氧自由基水平的变化特点。选择环孢霉素A（CsA）作为耐药逆转剂，分别采用MTT法、流式细胞术和免疫组织化学法分析CsA对人白血病耐药细胞系的毒性及逆转效果；用化学比色法检测逆转前后细胞内丙二醛（MDA）、超氧化岐化酶（SOD）和谷胱甘肽（GSH）的含量。结果表明：当CsA在4μg／ml以下时对HL-60／ADM无明显毒性作用，超过此浓度，其毒性呈剂量效应（P〈0．001）。当CsA浓度为0．5μg／ml时就有明显逆转作用，随着CsA剂量增加，逆转作用逐渐增强（P〈0．001），当CsA剂量达8μg／ml以上时对细胞存活产生明显的影响。流式细胞仪检测细胞内药物浓度发现，逆转耐药细胞系HL-60／ADM＋CsA细胞内阿霉素的浓度明显高于耐药细胞系HL-60／ADM。免疫组织化学检测结果显示，HL-60／ADM细胞膜上P—gP高表达，经CsA作用后P—gP表达下降。氧自由基测定结果显示：HL-60／ADM细胞经4μg／ml CsA作用72小时后细胞内SOD的活性与对照组相比显著降低（P〈0．001），细胞内的脂质过氧化产物MDA的含量与对照组相比有所升高（P〈0．05），抗氧化剂GSH的含量较对照组明显降低（p〈0．001）。结论：CsA能有效逆转HL-60／ADM的耐药性；逆转后细胞内氧自由基的含量升高，抗氧化剂的活性明显减低，过多的氧自由基可影响细胞的功能状态，导致细胞死亡。     

Effects of Intracellular Calcium Reduction by Dantrolene on Prevention/Treatment of Ischemic Stroke

BACKGROUND: It has been suggested that cerebral blood vessels and brain cells might depend more on intracellular calcium than extracellular calcium to modulate intracellular free calcium concentrations, [Ca(2+)](i). METHODS AND RESULTS: A potent intracellular calcium antagonist, dantrolene, was used to prevent the ischemic stroke induced in the rat model. It was found that treatment of rats with dantrolene at -1 hour and +1 hour after 60 minutes of ischemic insult prevented by the formation of cortical necrosis 98% and 85%, respectively. Further, the [Ca(2+)](i) of embryonic aorta cells was markedly reduced, and cAMP of the same cultured cells were significantly increased by dantrolene treatment. CONCLUSIONS: These results indicate that ischemic stroke is preventable by dantrolene through reduction of [Ca(2+)](i) and increase of cAMP.     

川芎嗪影响血管内皮生长因子诱导HL-60白血病细胞增殖的效应

背景:川芎嗪抑制血管内皮生长因子的表达,但对其诱导HL-60白血病细胞增殖是否有抑制效应尚需进一步实验.目的:观察川芎嗪对血管内皮生长因子诱导的白血病细胞HL-60细胞增殖的影响.设计:重复测量观察.单位:武汉科技大学医学院.材料:实验于2007-03/06在武汉科技大学医学院分子生物学实验中心完成.人白血病细胞系HL-60细胞购于上海细胞生物研究所.盐酸川芎嗪注射液为无锡市第七制药有限公司产品, 批号为011014,硫酸鱼精蛋白注射液购自上海第一生化药业公司,批号为010302, 免疫组化试剂盒购自博士德公司.方法:①取对数生长期人白血病细胞系HL-60细胞,加入100 μg/L 血管内皮生长因子,分别加入终浓度为1.5,15,150 mg/L川芎嗪实验培养基,以未加川芎嗪注射液的细胞为空白对照组,只含20 mg/L鱼精蛋白的细胞为阳性对照组,同时设立血管内皮生长因子对照组,细胞培养48 h后,采用MTT法检测HL-60细胞的生长抑制率.②川芎嗪影响HL-60细胞血管内皮生长因子蛋白表达实验:用终浓度为1.5,15及150 mg/L川芎嗪处理HL-60细胞,24 h后采用免疫组织化学法计算血管内皮生长因子蛋白阳性细胞表达率.主要观察指标:① HL-60细胞生长抑制率.②血管内皮生长因子蛋白表达情况.结果:① HL-60细胞生长抑制率:川芎嗪15, 150 mg/L作用血管内皮生长因子诱导的HL-60细胞吸光度值均低于血管内皮生长因子对照组,差异有显著性意义(P < 0.05).②血管内皮生长因子蛋白表达情况:川芎嗪作用HL-60细胞24 h后,血管内皮生长因子蛋白随川芎嗪给药浓度升高表达逐渐下调,呈一定依赖性,各川芎嗪浓度干预HL-60细胞血管内皮生长因子蛋白表达阳性细胞表达率与对照组比较差异均有显著性意义(P < 0.01).结论:川芎嗪可抑制血管内皮生长因子诱导HL-60细胞的增殖, 并下调血管内皮生长因子蛋白的表达.     

Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane.     

氯化锂对HL-60细胞增殖和分化与c-myc表达的影响

本实验用细胞培养技术观察了氯化锂对HL-60细胞增殖和分化的影响。不同浓度的氯化锂(5-20mmol/L)对HL-60细胞的液体悬浮培养细胞数以及集落形成和~3H-TdR掺入均呈剂量依赖式抑制;应用NBT还原试验发现氯化锂能诱导HL-60细胞分化。从氯化锂处理的HL-60细胞中提取总RNA,应用RT-PCR检测c-myc mRNA的表达结果表明经氯化锂处理的HL-60细胞c-myc表达,与未处理的HL-60细胞的c-myc比较有明显降低,说明c-myc在白血病细胞增殖、分化中起调控作用。     

白血病细胞系中抑癌基因PTEN启动子区甲基化状态检测及其去甲基化研究

目的 探讨抑癌基因PTEN表达沉默的机制及诱导PTEN表达对白血病细胞的作用.方法 应用甲基化特异性聚合酶链反应(Methylation specific PCR,MSP)检测白血病细胞系HL-60、Nalm-6、Raji、KG-1a、U937、NB4、K562中PTEN基因启动子区域甲基化状态;用甲基化转移酶抑制剂5-氮-2'-脱氧胞苷(5-Aza-CdR)处理白血病细胞,MSP检测甲基化状态的改变、RT-PCR检测PTEN mRNA水平的改变、瑞特染色观察细胞形态学改变、膜联蛋白V/碘化丙锭(Annexin V/PI)标记染色检测细胞凋亡.结果 检测的白血病细胞系中HL-60、Nalm-6、Raji、KG-1a、U937细胞PTEN基因显示超甲基化状态,而NB4和K562细胞显示低甲基化状态;5-Aza-CdR处理HL-60和Nalm-6细胞后,PTEN基因甲基化降低、mRNA表达水平则逐渐增高,并呈剂量依赖性,细胞出现凋亡现象.结论PTEN基因启动子区异常甲基化可能导致该基因转录表达失活或沉默,甲基化抑制剂可以诱导PTEN表达,并引起白血病细胞凋亡.     

Purine metabolism in myeloid precursor cells during maturation. Studies with the HL-60 cell line.

In studies with the human promyelocytic leukemia cell line HL-60, we defined changes in intermediary purine metabolism that appear to contribute to the regulation of terminal maturation in myeloid cells. When HL-60 cells were exposed to compounds that induce maturation, consistent alterations in purine metabolism were found to occur within 24 h of culture. Perturbation of guanosine nucleotide synthesis and decreases of up to 50% in intracellular guanylate pool sizes were associated with the induced maturation of these cells in response to diverse inducing agents. While immature HL-60 cells were observed to synthesize purine nucleotides by both de novo and salvage pathways, the activity of both pathways decreased in cells induced to mature, although the relative contribution of purine salvage increased. Moreover, incorporation of the salvage pathway precursor, [14C]hypoxanthine from the intermediate, inosine monophosphate (IMP), into guanylates was reduced by approximately 65% in induced HL-60 cells, reflecting decreased activity of both hypoxanthine phosphoribosyltransferase and IMP dehydrogenase. When various inhibitors of IMP dehydrogenase (mycophenolic acid, 3-deazaguanosine, and 2-beta-D-ribofuranosylthiazole-4-carboxamide) were evaluated for their effects upon HL-60 cells, each agent was found to induce the cells to mature morphologically and functionally. Like other inducers, these agents decreased HL-60 cell proliferation and caused the cells to acquire an ability to phagocytose opsonized yeast and reduce nitroblue tetrazolium. Each agent reduced intracellular guanosine nucleotide pool sizes and induced HL-60 cell maturation at micromolar concentrations. These observations suggest that the size of intracellular guanosine nucleotide pools, the biosynthesis of guanosine nucleotides, and the activity of IMP dehydrogenase may be central to the regulation of terminal maturation in myeloid cells.     

Retinoic acid. Inhibition of the clonal growth of human myeloid leukemia cells.

Vitamin A and its analogues (retinoids) affect normal and malignant hematopoietic cells. We examined the effect of retinoids on the clonal growth in vitro of myeloid leukemia cells. Retinoic acid inhibited the clonal growth of the KG-1, acute myeloblastic leukemia, and the HL-60, acute promyelocytic leukemia, human cell lines. The KG-1 cells were extremely sensitive to retinoic acid, with 50% of the colonies inhibited by 2.4-nM concentrations of the drug. A 50% growth inhibition of HL-60 was achieved by 25 nM retinoic acid. Complete inhibition of growth of both leukemia cell lines was seen with 1 microM retinoic acid. Exposure of KG-1 cells to retinoic acid for only 3-5 d was sufficient to inhibit all clonal growth. The all-trans and 13-cis forms of retinoic acid were equally effective in inhibiting proliferation. Retinal, retinyl acetate, and retinol (vitamin A) were less potent inhibitors. Clonal growth of the human K562 and mouse M-1 myeloid leukemic cell lines was not affected by 10 microM retinoic acid. Retinoic acid also inhibited the clonal growth of leukemia cells from five of seven patients with acute myeloid leukemia. Retinoic acid at concentrations of 5 nM to 0.3 microM inhibited 50% clonal growth, and 1 microM retinoic acid inhibited 64-98% of the leukemic colonies. The inhibition of clonal growth of KG-1 and HL-60 cell lines and of leukemic cells from two patients was not associated with the presence of a specific cytoplasmic retinoic acid-binding protein. Our study suggests that retinoic acid may prove to be effective in the treatment of human myeloid leukemia.     

In vitro effects of ciprofloxacin and pefloxacin on growth of normal human hematopoietic progenitor cells and on leukemic cell lines

The in vitro effect of ciprofloxacin and pefloxacin on growth of normal hematopoietic progenitor cells and on leukemic cell lines was investigated. Ciprofloxacin and pefloxacin caused dose-dependent inhibition of colony formation both from normal bone marrow cells and from the leukemic line K-562 cells. This inhibition exerted by ciprofloxacin and pefloxacin was statistically significant at concentrations of 25 micrograms/ml and above. Ciprofloxacin appeared to be the most potent inhibitor of colony formation among the antimicrobial agents tested. Although the inhibitory effect of pefloxacin on normal hematopoietic stem cells was similar to that of cefazolin and chloramphenicol, the inhibitory effect of pefloxacin on leukemic cells was more prominent than that of cefazolin and chloramphenicol. In a proliferation assay in liquid culture of the cell line HL-60, ciprofloxacin and pefloxacin caused a dose-dependent inhibition of cell proliferation. Both drugs failed to induce cellular differentiation, as assessed by the nitrogen blue tetrazolium dye reduction assay. In therapeutic concentrations no cumulative toxic effect of the combination of ciprofloxacin with cytosine-arabinoside, vincristine, actinomycin D and doxorubicin on colony formation by HL-60 cells was observed. It is concluded that ciprofloxacin does not exert in vitro inhibitory effect on human leukemic cells when assayed at concentrations of less than or equal to 5 micrograms/ml. However, at concentrations of 25 and 50 micrograms/ml of ciprofloxacin alone and in combination with several antineoplastic agents exerts an inhibitory effect on colony formation.