HL-60细胞及其耐药细胞株HL-60/ADM超弱发光的检测

目的:探讨人急性早幼粒白血病细胞系(HL-60)和其耐药株抗阿霉素人急性早幼粒白血病细胞系(HL-60/ADM)的超弱发光与耐药性的关系。方法:应用MTT比色法、流式细胞术、细胞生长曲线和免疫组化染色法,检测HL-60细胞、HL-60/ADM细胞对阿霉素(ADM)的敏感性、细胞周期变化及P-糖蛋白(P-gp170)的表达。用IFFM-D型流动式化学发光仪检测HL-60细胞及HL-60/ADM细胞的超弱发光强度。结果:HL-60/ADM细胞对ADM的敏感性明显低于亲本HL-60细胞;HL-60/ADM细胞中G0/G1期的细胞为44·80%±1·97%,G2/M期的细胞为9·90%±0·27%,较其亲本细胞增多,S期的细胞为45·30%±1·93%,较其亲本细胞减少(P<0·05);HL-60/ADM细胞倍增时间是HL-60细胞的1·8倍,P-gp170的表达呈阳性,HL-60细胞呈阴性。在1×10-4mol/L鲁米诺及3mL/L双氧水条件下,密度分别为8×107/L、1×108/L、1·26×108/L及2·73×108/L的HL-60/ADM细胞超弱发光强度低于HL-60细胞(P<0·05)。结论:HL-60/ADM细胞对ADM的敏感性降低,细胞生长延缓,P-gp170表达呈阳性等均提示HL-60/ADM细胞出现了耐药性;且随着HL-60/ADM细胞耐药性的产生其超弱发光强度较其亲本细胞HL-60降低,提示细胞超弱发光强度明显降低可作为细胞耐药性产生的一项指标。     

The degranulation response in differentiated HL-60 cells

HL-60 cells differentiate into mature granulocytes in response to treatment with a variety of chemical agents. Such HL-60 cell derived granulocytes display many of the properties associated with their peripheral blood counterpart. In this study we have investigated the development of the degranulation response in dimethylsulfoxide (DMSO) or retinoic acid differentiated HL-60 cells over a six day period. The release of a number of enzymes in response to stimulation by a variety of agents was examined. Soluble aggregated IgG (SAIgG) stimulated the release primarily of elastase from HL-60 derived granulocytes with little or no release of other granule enzymes, in particular myeloperoxidase. This contrasted to what was seen when peripheral blood granulocytes were used. The lack of myeloperoxidase release was not due to the parallel release of enzyme inhibitors or failure of the stimulus to bind to the cells. Neither was it due to variations in the kinetics of enzyme release or the presence of myeloperoxidase and elastase in discrete sub-populations of HL-60 cells. When other stimuli such as fMet-Leu-Phe, A23187, or phorbol myristate acetate (PMA) were used a relatively normal degranulation response was seen. Thus, the degranulation response in granulocytes derived from HL-60 cells appears relatively normal when a range of commonly used stimuli are used but is impaired when aggregates of IgG are used.     

IL-24基因修饰增强CIK细胞对HL-60细胞的细胞毒作用

目的:研究IL-24基因修饰的CIK细胞与同源树突状细胞共培养后对白血病细胞的杀伤作用及其机制.方法:从健康人外周血单个核细胞中常规诱导DC和CIK 细胞,电穿孔法将IL-24基因导入CIK细胞中(获得细胞为CIK-IL24),RT-PCR 和ELISA法检测CIK细胞中IL-24基因的表达,FCM和ELISA法检测转基因前后CIK表型及分泌细胞因子能力的变化,将CIK 细胞和同源DC共培养,FCM法检测共培养的DC-CIK细胞对HL-60细胞细胞毒活性的变化.结果:通过电穿孔法成功将IL-24基因导入CIK细胞,与对照组相比,转IL-24基因后CIK细胞中CD3~+、CD3~+CD56~+细胞的比例无明显改变,CD4~+CD25~+细胞比例显著下降.IL-24可上调CD3+CD56+细胞表面粘附分子CD54、CXCR4的表达,转染IL-24基因后CIK分泌TNF-α和IFN-γ的能力显著增强,与DC共同作用HL-60细胞时转染IL-24基因后的CIK细胞细胞毒活性明显增强.结论:通过IL-24基因修饰,明显增强了CIK细胞对HL-60细胞的杀伤能力,其机制与IL-24促进CIK分泌TNF-α、IFN-γ,上调CIK细胞表面粘附分子的表达,减少CD4~+CD25~+调节性T细胞比例等密切相关.     

Changes in susceptibility to Fas-mediated apoptosis during differentiation of HL-60 cells

The Fas-mediated pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the susceptibility to Fas-mediated apoptosis of HL-60 cells treated with differentiation-inducing factors such as dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1alpha, 25 dihydroxyvitamin D3 (VD3). Although the expression of Fas antigen (Ag) and its mRNA showed a marked increase in HL-60 cells with cell differentiation, that of Bcl-2 protein and its mRNA revealed the reverse. The expression of caspase proteins such as caspases-3 and -8 was also enhanced during cell differentiation. DNA fragmentation, annexin V binding, and caspase activities increased in differentiated HL-60 cells with the addition of anti-Fas Ag antibody. These findings were more clearly demonstrated in DMSO- or RA-induced neutrophil-like cells than in VD3-induced monocyte-like cells. Therefore, susceptibility to Fas-mediated apoptosis showed an increase with differentiation of HL-60 cells, especially in the neutrophil lineage. These results suggest that the difference of susceptibility to Fas-mediated apoptosis among cell populations depends on the expression of Fas Ag, Bcl-2, and caspases. Cell maturation and susceptibility to Fas-mediated apoptosis may be linked in hematopoietic cells.     

维生素K3对HL-60细胞凋亡的诱导作用

目的:观察维生素K₃(VK₃)对HL-60细胞凋亡的诱导作用。方法:通过形态学、DNA电泳、Annexin V标记法检测VK₃作用后HL-60细胞的凋亡发生情况。结果:2、5、10、20 μmol/L的VK₃作用于HL-60细胞48h后凋亡发生率分别为8.76%、9.7%、18.54%、75.4%;10 μmol/L的VK₃作用于HL-60细胞24、48、72、96 h后凋亡发生率分别为11.35%、18.54%、21.22%、37.54%。结论:VK₃能诱导HL-60细胞发生凋亡,并具有一定的量效和时效关系。     

Factors influencing basophilic differentiation of HL-60 cells

Lineage-specific hematopoietins apparently act in concert with multipotent factors in an orderly sequence of growth and differentiation. We have used the human acute promyelocytic leukemia cell line HL-60 to examine basophilic differentiation, using radioenzymatic assay of histamine content as an end point. Recombinant human interleukin 1 (rhIL-1), rhIL-2, rhIL-4, and recombinant human alpha and gamma interferons did not stimulate basophilic differentiation either in the presence or absence of sodium butyrate, an important cofactor for induction of differentiation. In contrast, rhG-CSF (granulocyte colony-stimulating factor), rhGM (granulocyte-macrophage) CSF, rhIL-3, rhIL-5, nerve growth factor, conditioned medium (CM) from the hairy T cell leukemic line Mo, and nasal polyp epithelial CM stimulated significant increases in histamine content in HL-60 cells at day 5 in vitro. GM-CSF did not account for all of the basophilic differentiating activity in Mo-CM. The data suggest that a unique, lineage-specific, basophilic cell differentiation factor is produced by T cells and point to the possible diagnostic and therapeutic relevance of in situ hematopoietic mechanisms in human respiratory disease.     

双歧杆菌脂磷壁酸对HL-60细胞端粒酶活性的影响

目的 探讨双歧杆菌表面分子脂磷壁酸（ＬＴＡ）对体外培养的ＨＬ－６０白血病细胞端粒酶活性的影响。方法 采用ＰＣＲ－ＥＬＩＤＡ法检测经ＬＴＡ处理前后的ＨＬ－６０白血病细胞株端粒酶活性的改变。结果 经ＬＴＡ处理后,ＨＬ－６０白血病细胞的生长受到抑制,端粒酶活性明显降低。结论 双歧杆菌ＬＴＡ对ＨＬ－６０白血病细胞具有生长抑制作用,其抗肿瘤作用的机理可能与抑制肿瘤细胞的端粒酶活性有关。     

茶多酚在体外诱导HL-60细胞的凋亡

目的：寻找新的肿瘤细胞凋亡诱导剂。方法：选用茶的主要活性成分茶多酚,采用噻唑蓝（ＭＴＴ）还原法,ＤＮＡ凝胶电泳以及透射电镜技术,在体外观察了茶多酚对人急性早幼粒白血病细胞（ＨＬ－６０）凋亡的影响。结果：被２５０ｍｇ／Ｌ茶多酚作用５ｈ后ＨＬ－６０细胞进行凝胶电泳呈现出典型的ＤＮＡ条带,透射电镜下看到凋亡小体,茶多酚对ＨＬ－６０细胞凋亡的诱导活性平行于它的细胞杀伤作用。结论：在体外培养条件下,茶多酚可诱导ＨＬ－６０细胞凋亡     

达努塞替对HL-60细胞抗肿瘤作用机制的研究

目的:探讨丝氨酸/苏氨酸激酶抑制剂达努塞替对人急性髓细胞白血病(AML)细胞株HL-60的细胞周期阻滞、自噬和凋亡的影响。方法:用MTT法检测达努塞替对HL-60细胞的生长抑制作用;用流式细胞术检测相同时间不同浓度药物组与同一浓度不同时间组的细胞周期、细胞凋亡及细胞自噬;用Western blot方法检测细胞周期相关蛋白CDK1/CDC2、CDK2、cyclin B1、P21Waf1/Cip1、P27Kip1和P53,凋亡相关蛋白Bcl-x L、Bcl-2、Bax、PUMA、cleaved caspase-3及cleaved caspase-9以及自噬相关蛋白p-PI3K、PTEN、p-Akt、p-m TOR、Beclin 1、LC3-I和LC3-II的水平。用共聚焦显微镜检测同时间不同浓度药物组细胞的自噬情况。结果:达努塞替能明显抑制HL-60细胞的活力,诱导细胞周期G2/M期阻滞,可以通过线粒体caspase-3途径诱导细胞凋亡,同时可以通过抑制PI3K/Akt/m TOR信号通路诱导细胞自噬。结论:达努塞替可通过抑制作为染色体伴侣蛋白且在有丝分裂中调控染色质复制与分离的极光激酶A/B有效抑制HL-60细胞生长,引起细胞周期阻滞、凋亡和自噬发生,可能是AML临床治疗具有前景的抗肿瘤靶点。     

Histamine-induced differentiation of eosinophilic subclone of HL-60 cells

Inflammation Research -     

Cryopreservation of differentiated HL-60 cells for pyrogen testing

All-trans retinoic-acid (ATRA) differentiated HL-60 cells can be used to detect pyrogens such as bacteria, bacterial components, yeasts and fungi. Differentiated HL-60 cells obtain neutrophil like characteristics and if stimulated the differentiated HL-60 cells produce reactive oxygen species in a dose dependent manner. Culturing and differentiation of cell lines are time consuming activities and require suitable facilities; cryopreservation of pre-differentiated cells could provide the basis for an easily distributable pyrogen testing kit. Cryopreservation of granulocytes has proven to be very complicated and neutrophils are especially difficult to cryopreserve, most likely due to their large degree of granulation. Here we present evidence that HL-60 cells can be differentiated with ATRA and subsequently cryopreserved. Upon thawing the cells retain their ROS producing capabilities and reactivity towards pyrogens. Further, the cells retain their ability to react dose dependently towards lipopolysaccharide (LPS), lipoteichoic acid (LTA) and zymosan. At pathophysiologically relevant concentrations of LPS, LTA and zymosan the cells retain full reactivity for at least two months when stored in liquid nitrogen. In conclusion, ATRA differentiated HL-60 cells are cryopreservable and retain reactivity upon thawing. It is therefore possible to produce an in-vitro in-house pyrogen test kit for medicines and related products.     

黄芩苷对HL-60白血病细胞的诱导分化作用

目的： 观察中药单体黄芩苷(baicalin) 对人髓系白血病（AML）细胞株HL-60细胞的诱导分化作用。方法: 应用细胞形态学方法、细胞克隆形成试验、流式细胞术分析和NBT还原实验检测黄芩苷诱导HL-60细胞分化的能力。结果: 黄芩苷可诱导HL-60细胞向成熟阶段分化，低浓度黄芩苷可显著抑制HL-60细胞克隆的形成；HL-60细胞经黄芩苷处理后CD11b表达显著增高，CD33表达显著降低；NBT还原实验示黄芩苷处理组的分化成熟细胞阳性率明显高于未加药组。结论: 黄芩苷具有诱导HL-60白血病细胞向成熟粒细胞分化的作用。     

Increased intracellular cAMP renders HL-60 cells resistant to cytotoxicity of taxol.

The treatment of advanced cancers with paclitaxel (taxol) is hindered by the development of drug resistance. Resistance to taxol is known to be associated with multidrug resistance (MDR) and a mutation affecting either the alpha- or beta-subunit of tubulin. In this study, we demonstrated that an intracellular cAMP level may also play an important role in resistance to taxol in HL-60, acute promyelocytic leukemia cells. Exposure of HL-60 cells to various doses of taxol for 18 hr resulted in cell death. However, pretreatment of the cells with cAMP analogs such as N6:O2-dibutyl cAMP (Db-cAMP), 8-(4-chlorophenylthio) cAMP (CPT-cAMP) and 8-bromo-cAMP (8-Br-cAMP) or an intracellular cAMP elevating agent such as forskolin apparently rendered HL-60 cells more resistant to taxol, but not with dimethyl sulfoxide (DMSO) or retinoic acid (RA), well known differentiating agents. To investigate whether protein kinase A (PKA) activated by an increase in intracellular cAMP level could be involved in increased taxol resistance of the cells, we examined the effects of PKA inhibitors, including H-89 and KT5720, on taxol resistance induced by Db-cAMP. The PKA inhibitors significantly abolished Db-cAMP-induced taxol resistance. These results suggest that cAMP analogs may render tumor cells more resistant to taxol via PKA activation.     

AKT-mediated regulation of polarization in differentiated human neutrophil-like HL-60 cells

Objectives

Neutrophil polarization is critical for the inflammatory response. AKT is a serine/threonine protein kinase and has been implicated in cell migration. However, it is not completely clear whether AKT affects neutrophil polarization. In this study, we tested the hypothesis that AKT regulates the polarization of neutrophil-like differentiated HL-60 cells (dHL-60) in response to fMLP.

Methods

HL-60 cells were differentiated into dHL-60 by incubation in medium containing 1.3?% DMSO for up to 6?days. Polarization of dHL-60 cells and primary human neutrophils were measured by Zigmond chamber. Phospho-Akt was analyzed by immunofluorescence and Western blot analysis. F-actin polymerization was detected by Rhodamine-Phalloidine staining. Rac2 activation was evaluated using GST Pull-down assay.

Results

We found that changes in the rate of cell polarization were consistent with the changes in AKT phosphorylation levels during HL-60 cell differentiation in response to fMLP. Moreover, cell polarization and AKT phosphorylation were reduced in fMLP-stimulated dHL-60 cells pretreated with the PI3 kinase inhibitors or the AKT inhibitors, which was confirmed in the primary human neutrophils. The AKT inhibitors altered fMLP-induced F-actin polymerization. Rac2 GTPases was also decreased by the AKT inhibitors in fMLP-stimulated dHL-60 cells.

Conclusion

This study demonstrates that AKT activation plays a crucial role in dHL-60 cell polarization.     

天花粉蛋白诱导人类白血病细胞株HL-60细胞凋亡的研究

目的:研究单链核糖体失活蛋白天花粉蛋白(TCS)诱导人类白血病细胞株HL-60细胞发生凋亡的作用及放线菌酮(CHX)对此作用的影响。方法:采用流式细胞术分析及荧光显微镜观察研究TCS诱导HL-60细胞发生凋亡的情况。结果:流式细胞术分析表明TCS能够诱导HL-60细胞发生明显的凋亡现象,TCS(20mg/L)处理48h细胞凋亡百分率为48.7%±2.3%(x±s),明显高于对照组的凋亡率(6.3%±1.0%)(P＜0.05),而CHX(5mg/L)相同条件下诱导的凋亡率为65.3%±3.9%。TCS诱导的凋亡现象进一步为荧光显微镜的观察及DNA凝胶电泳所证实,TCS处理的细胞中有许多细胞呈现典型的凋亡细胞核形态改变,如染色体凝缩、核碎裂等;DNA凝胶电泳显示TCS处理的细胞呈典型的梯级格局。进一步研究表明预先以低浓度CHX(0.2mg/L)处理可显著加强TCS的作用,而在这个浓度下单独CHX并不诱导明显的细胞凋亡。TCS诱导HL-60细胞的凋亡呈时间和剂量依赖关系。结论:TCS能够诱导HL-60细胞发生明显的凋亡,CHX可加强这种作用。这些结果提示TCS诱导的凋亡不依赖于新的蛋白质合成。     

Actin distribution patterns in HL-60 leukemia cells treated with etoposide

Localization of actin was studied in HL-60 leukemia cells after treatment with the anticancer agent etoposide for 3 days in a range of concentrations (0.02-200 microM). Significant changes in morphology of the cells and F-actin distribution patterns labelled with TRITC-phalloidin occurred only after treatment with 100 and 200 microM etoposide. In comparison with control cells, the number of cells decreased, cells were larger and almost all treated cells had irregular surfaces with lamellipodia. F-actin was distributed in a punctate pattern throughout the cytoplasm after treatment. In some treated cells, fluorescence appeared as a bright haze, whereas in other cells it formed a network. Treated cells also showed bright fluorescence at their periphery. Immunogold labelling of actin was observed in cells whether or not treated with etoposide. Labelling was found in the nucleus and also in the cytoplasm. At the ultrastructural level, cells treated with 100 and 200 microM etoposide showed increased positivity for actin in relation with blebbing, margination of nuclear chromatin and bodies containing recognizable nuclear fragments. These findings indicate that alterations in expression of actin in HL-60 cells after treatment with etoposide is dose-dependent and related with apoptosis.     

米托蒽醌对HL-60细胞的促凋亡效应

目的 :探索米托蒽醌 (MTN)促HL 6 0细胞凋亡效应及其机理。方法 :取对数生长期HL 6 0细胞 ,不同浓度MTN作用不同时间后 ,利用MTT法、透射电镜、DNA电泳、流式细胞术观察MTN对HL 6 0细胞的抑制效应、促凋亡效应及凋亡相关基因Bcl 2、Bax蛋白表达情况。结果 :(1)毫微克级的MTN对HL 6 0细胞有明显的抑制效应 ,且效应具有时间、剂量依赖关系。(2 )MTN作用后的HL 6 0细胞透射电镜观察胞膜完整、出现核碎裂、凋亡小体等形态学特征。DNA电泳出现 2 0 0bp左右的梯形条带。 (3)Bcl 2、Bax蛋白表达 :MTN作用于HL 6 0细胞相同时间 ,Bcl 2蛋白及Bcl 2蛋白与Bax蛋白比值 (Bcl 2 Bax)随MTN作用时间延长而下降。结论 :MTN具有诱导HL 6 0细胞凋亡作用 ,此过程中伴Bcl 2下调和Bcl 2 Bax的降低。提示MTN可能通过Bcl 2途径诱导HL 6 0细胞凋亡