运用蛋白质组学研究三氧化二砷诱导K562细胞凋亡过程中核基质蛋白的变化

目的:探讨三氧化二砷(As2O3)在体外对慢性粒细胞性白血病细胞系K562细胞核基质蛋白的影响. 方法:用DNA梯度电泳观察As2O3作用后K562细胞凋亡的情况.单向、双向电泳观察As2O3对K562细胞核基质蛋白分布影响. 结果:2.5 μmol/L As2O3处理72 h后,DNA电泳已能检测到发生凋亡的K562细胞,但早在As2O3处理48 h时, 即能观察到核基质蛋白分布的改变,此时尚不能观察到凋亡特征性的DNA片段梯形条带.双向电泳可检测出200多个核基质蛋白点,其中约18个点与As2O3处理相关. 结论:As2O3对K562细胞的影响是时间和剂量依赖性的.双向电泳检测As2O3对K562核基质蛋白分布的影响较DNA梯度电泳检测更为敏感.核基质蛋白成分可能是砷作用的靶蛋白,并且可能是药物发挥抗癌作用的关键点之一.     

三氧化二砷诱导慢性髓系白血病细胞G2/M周期停滞及其机理

目的 研究三氧化二砷对慢性髓系白血病细胞的体外作用特点及其机理。方法 将三氧化二砷与慢性髓性白血病细胞系K562作用后，测定细胞的生长曲线及活性，流式细胞测量术检测细胞凋亡和细胞周期分布的改变；同时，采用RT-PCR方法检测药物作用前后细胞周期相关基因表达的变化。结果 ①三氧化二砷明显抑制K562细胞的生长，其作用呈时间和浓度依赖性；②三氧化二砷诱导K562细胞凋亡的作用较弱，但可明显诱导K562细胞在G2／M期停滞，此效应也呈时间和浓度依赖性；③三氧化二砷作用后，细胞周期抑制基因p57表达明显上调，而细胞周期促进基因cyclinB的表达受到抑制。结论 三氧化二砷能有效抑制慢性髓系白血病细胞的生长，其机理主要为诱导细胞G2／M期周期停滞；该效应与细胞周期相关基因的表达变化有关。     

千层纸素诱导人慢性粒细胞白血病K562的凋亡作用

探讨千层纸素对人慢性粒细胞白血病细胞K562的凋亡诱导作用及其可能机制。采用MTT法检测细胞存活率;电镜下观察细胞超微结构的改变;DAPI染色观察细胞凋亡;流式细胞仪检测细胞凋亡率;Western blot法分析凋亡相关蛋白caspase 9、survivin及ERK1/2的表达变化情况。结果表明千层纸素呈浓度依赖地抑制K562细胞的增殖作用,诱导K562细胞发生凋亡,上调 K562细胞中cleaved-caspase 9蛋白,下调survivin、pro-caspase 9及p-ERK1/2蛋白的表达水平。千层纸素可诱导K562凋亡,这可能与下调survivin蛋白表达,激活caspase 9蛋白,抑制ERK信号通路有关。     

Molecular Mechanism of Hydroxyurea Enhances K562 Cell Apoptosis Induced by Tumor Necrosis Factor-related Apoptosis-inducing Ligand

:Objective To explore the molecular mechanism via which the chemotherapeutic drug hydroxyurea (HU) enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Methods Chronic myelogenous leukemia-derived K562 and SVT-35 cells were treated with recombinant soluble TRAIL (rsTRAIL) alone or combined with HU for a time course, and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl-2H-tetrazolium-phenazine methosulphate assay. Western blot was performed to analyze the activation of apoptosis-related protein kinases and the expression of apoptosis inhibitor molecules. Results The survival rates of SVT-35 and K562 cells treated with 1 μg/ml rsTRAIL for 24 hours were 32% and 93%, respectively. HU significantly increased the sensitivity of K562 cells to rsTRAIL cytotoxicity. Combination of rsTRAIL and HU resulted in the phosphorylation of rat sarcoma (RAS), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase and in the significant reduction of apoptosis-inhibited molecule Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 in K562 cells. Conclusions HU enhanced K562 cell sensitivity to rsTRAIL is mediated by Ras-MEK-ERK signaling pathway. Expression of antiapoptotic proteins cellular Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 is also down-regulated during this process. These results may through light on the therapeutic study of human chronic myelogenous leukemia.     

Arsenic trioxide inhibits p-glycoprotein expression in multidrug-resistant human leukemia cells that overexpress the MDR1 gene

Objective To investigate the effects of arsenic trioxide (As2O3) on the apoptosis and p-glycoprotein (P-gp) expression of multidrug-resistant human leukemia cells.Methods Human multidrug-resistant leukemia cell line K562/ADM overexpressing the MDR1 gene, was used as the target cells. The cell proliferating activity was assessed using the MTT colorimetric assay. Cytomorphology was investigated under light, confocal and electron microscopes. DNA fragmentation was examined using agarose gel electrophoresis, while p-gp expression, cell cycle status and sub-G1 cells were determined using flow cytometry.Results Zero point five to 20 μmol/L As2O3 inhibited the proliferation of K562/ADM cells, and K562/ADM cells were more sensitive to As2O3 than the parental K562 cells. As2O3-induced apoptosis of K562/ADM cells was determined by the observance of typical morphological changes and the appearance of DNA ladder and sub-G1 cell populations. As2O3 significantly inhibited the P-gp expression of K562/ADM cells, and synergistically enhanced the sensitivity of the drug-resistant cells to adriamycin.Conclusions As2O3 induces growth-inhibition and apoptosis, down-regulates P-gp expression and exerts a synergistic effect in combination with adriamycin in multidrug-resistant leukemia cells.     

小分子酪氨酸激酶抑制剂AG-490对人慢性髓系白血病急变细胞株K562细胞增殖凋亡的影响

目的探讨酪氨酸激酶抑制剂AG-490对人慢性髓系白血病急变细胞株K562细胞的增殖及凋亡的影响。方法采用MTT法观察AG-490在体外对人慢性髓系白血病急变细胞株K562细胞增殖的影响,光学显微镜下观察AG-490对K562细胞形态的变化,流式细胞术检测AG-490对K562细胞凋亡及细胞周期的影响,逆转录-聚合酶链反应（RT-PCR）及实时荧光定量PCR技术检测AG-490作用后K562细胞bcr/abl融合基因及凋亡相关基因c-myc mRNA的表达水平。结果 AG-490可明显抑制K562细胞的增殖,并随药物作用时间的延长和浓度的增加,抑制作用越明显（P〈0.05）;AG-490可使细胞周期阻滞于G1期,促使其凋亡（P〈0.05）。AG-490作用于K562细胞48 h后bcr/abl融合基因的表达无明显改变（P〉0.05）,而c-myc的表达水平明显降低（P〈0.05）。结论 AG-490可能通过阻断JAK2信号通路而下调c-myc的表达,使K562细胞阻滞于G1期,从而抑制K562细胞增殖,促使其凋亡。     

小干扰RNA对白血病细胞K562的生长和蛋白表达的影响

目的:检测小干扰RNA对慢性粒细胞白血病细胞系K562的影响。方法:脂质体转染细胞,MTT实验检测转染后的细胞活性,并绘制生长曲线,用Western blot检测p210蛋白表达水平的变化,并用Annexin-V-FITC法检测细胞凋亡水平。结果:与对照组相比,有2条小干扰RNA序列可以有效地降低细胞活性,诱导细胞凋亡并降低p210蛋白的表达水平。结论:小干扰RNA可抑制K562细胞活性,降低融合蛋白表达水平并诱导细胞,有望成为治疗慢性粒细胞白血病的有效方法。     

氯化汞对K562和HEL细胞株作用的实验研究

为探讨中药水银（Ｈｇ）和经粉（ＨｇＣｌ）的抗肿瘤作用,以三氧化二砷（Ａｓ２Ｏ３）及阿糖胞苷（Ａｒａ－ｃ）为对照,应用细胞形态学、流式细胞术、ＭＴＴ法和药物反应曲线等方法观察３种药物对Ｋ５６２和ＨＥＬ细胞株的作用。结果表明,ＨｇＣｌ２和Ａｓ２Ｏ３均能诱导Ｋ５６２细胞凋亡,而Ａｒａ－ｃ无此作用。ＨｇＣｌ２可使Ｋ５６２的Ｇ０／Ｇ１增高,揭示激活Ｐ５３基因可能是氯化汞诱导凋亡机制之一;ＨｇＣｌ２和Ａｓ２Ｏ     

三氧化二砷诱导白血病多药耐药细胞凋亡的形态学改变

目的 系统观察三氧化二砷（As2O3)诱导白血病多药耐药细胞凋亡时的形态学特征。方法 用As2O3诱导白血病多药耐药细胞K562/ADM细胞凋亡，光镜，激光共聚焦显微镜（Confocal)和电子显微镜观察凋亡细胞的形态学变化。结果 As2O3诱导K562/ADM细胞凋亡，形态学上出现典型的凋亡特征性改变。发生凋亡的细胞可被邻近的未凋亡细胞通过吞噬等方式清除，且正常细胞吞噬凋亡细胞后自身出现某些凋亡的形态学改变。结论 体外诱导凋亡的白血病耐药细胞可被邻近的同类细胞吞噬清除。这种行为可能引发同类“吞噬”细胞自身发生继发性凋亡。     

白血病细胞株耐药与核因子κB蛋白质诱导表达的关系

目的:探讨慢性粒细胞性白血病细胞K562与其耐药株K562/ADR细胞中IκB-α和NF-κB的表达,以及三氧化二砷(As2O3)诱导白血病细胞凋亡的关系.方法:应用不同浓度的As2O3诱导K562和K562/ADR细胞凋亡,以Western-blot免疫印迹电泳检测NF-κB、IκB-α的表达,流式细胞术检测细胞凋亡及分析IκB-α的阳性表达率变化.结果:As2O3诱导K562/ADR细胞凋亡率明显低于K562细胞,当As2O3浓度为1 μmol/L时,两种细胞凋亡率分别是(6.33±1.51)%、(13.25±1.83)%,当As2O3浓度增高到4 μmol/L时,细胞凋亡率则分别为(8.00±1.47)%、(50.56±8.62)%(P＜0.05).在K562细胞胞浆中,IκB-α阳性表达率则由(88.07±0.99)%减少到(49.21±0.95)%(P＜0.01),胞核中P65量逐渐增加;而K562/ADR细胞中无明显变化.结论:As2O3可诱导K562细胞凋亡,伴有IκB-α的降解及NF-κB的激活;K562/ADR细胞NF-κB表达增加,对As2O3诱导的反应无明显改变.     

痰热清注射液对慢性髓系白血病K562细胞增殖的抑制作用

目的观察痰热清注射液对慢性髓系白血病K562细胞增殖的抑制作用。方法将痰热清注射液倍比稀释成1：2、1：4、1：8、1：16、1：32、1：64、1：128、1：256、1：512共9个浓度组,分别处理增殖期慢性髓系白血病K562细胞,镜下观察不同时间点各浓度组细胞生长情况,应用CCK-8法检测细胞增殖能力,绘制生长曲线,计算抑制率和半数抑制浓度（IC50）。结果痰热清注射液能明显抑制K562细胞增殖,细胞倍增时间为24h,1：2至1：8稀释浓度组的细胞毒性大,细胞基本死亡;抑制作用呈剂量和时间依赖性,IC50约为1：55稀释浓度。结论痰热清注射液对K562细胞具有抑制作用,这为临床应用痰热清注射液治疗血液肿瘤并发感染提供了实验基础。     

三氧化二砷对肝癌细胞系HepG2生长及核基质蛋白的影响

目的 探讨三氧化二砷对人肝癌细胞系ＨｅｐＧ２的生长及核基质蛋白的影响。方法 应用生长指数分析和高分辨ＳＤＳ－聚丙烯酰胺交双向电泳比较用Ａｓ２Ｏ３处理后的ＨｅｐＧ２细胞主核基质蛋白的改变。结果 Ａｓ２Ｏ３抑制ＨｅｐＧ２的生长,双向电泳显示经Ａｓ２Ｏ３处理后ＨｅｐＧ２细胞内出现新的核基质蛋白,并且该蛋白是细胞特异性的。结论 核基质蛋白是某些抗癌药物的作用靶子,Ａｓ２Ｏ３抑帛 癌细胞的生长可望有用于临床     

Herbimycin A enhances apoptotic effect of chemotherapeutic drugs on K562 cells

OBJECTIVES: To explore the anti-apoptotic mechanism and the effective apoptosis-inducing method in chronic myelogenous leukemia (CML) cells. METHODS: K562 cell line was used to observe the effect of combination of herbimycin A (HMA), a tyrosine kinase inhibitor, and chemotherapeutic agents on the induction of apoptosis. RESULTS: HMA or chemotherapeutic agents could inhibit the proliferation but not significantly induce apoptosis of K562 cells. However, HMA significantly enhanced apoptosis when combined with chemotherapeutic agents. Addition of sulfhydryl compound to the cultures to conjugate HMA completely abrogated this enhancing effect on K562 cells. CONCLUSIONS: HMA increases the sensitivity of CML cells to chemotherapeutic agents by inactivating tyrosine kinase activity. It is promising that combination of HMA with conventional chemotherapeutic drugs may be a new strategy in the treatment of CML.     

As2O3治疗白血病的机制探讨

目的 探讨三氧化二砷（As2O3）治疗白血病的作用机制。方法 在K562细胞中表达带3蛋白膜段结构域，经Western Blot检定其表达；通过离子转运测定验证其生物学活性；应用As2O3诱导细胞凋亡，通过电镜观察、线粒体膜电位测定和凋亡片断DNA的提取，观察As2O3对表达带3蛋白膜段结构域的K562细胞凋亡的影响。结果 表达带3蛋白膜段结构域的K562细胞的凋亡特征比未表达带3蛋白的K562细胞明显。说明表达带3蛋白膜段结构域的K562细胞对As2O3更敏感。结论 带3蛋白参与了凋亡的过程，为探讨As2O3治疗白血病的机制提供一条新的线索。     

Genistein 对K562细胞生长及CyclinD1基因和bcr-abl融合基因表达的影响

目的 研究Genistein抑制K562细胞生长的机制及对CyclinD1基因和bcr-abl融合基因表达的影响。方法 用RT－PCR方法检测Genistein作用于K562细胞前后CyclinD1基因和bcr-abl融合基因的mRNA表达变化,用免疫荧光、流式细胞仪和电镜检测CDK4蛋白的表达、细胞周期及凋亡。结果 K562细胞中CyclinD1基因、bcr-abl融合基因的mRNA和CDK4蛋白表达阳性,用Genistein与K562细胞共同孵育后,bcr-abl融合基因的mRNA表达阴性,CyclinD1的mRNA和CDK4蛋白表达下降,细胞阻滞于G2／M期,K562细胞出现凋亡。结果 bcr-abl融合基因、CyclinD1基因和CDK4蛋白在K562细胞中高表达,Genistein能使K562细胞生长受抑,诱导其调亡,抑制bcr-abl基因的mRNA表达,并使CyclinD1基因的mRNA和CDK4蛋白表达下降,表明bcr-abl融合基因、CyclinD1基因和CDK4对慢粒发展存在内在关系。     

Antitumor effect of betulinic acid on human acute leukemia K562 cells <Emphasis Type="Italic">in vitro</Emphasis>

The effects of betulinic acid (BA), a pentacyclic lupane-type triterpene, on the cell viability, cell cycle and apoptosis in human leukemia K562 cells were investigated. The effects of BA on the growth of K562 cells were studied by MTT assay. Apoptosis was assayed through Annexin V/propidium iodide (PI) double-labeled cytometry. The effects of BA on the cell cycle of K562 cells were studied by a PI method. The expression of Bax and capase-3 was detected by using Western blot. The results showed that BA was ...     

枸杞多糖对人白血病K562细胞株凋亡作用的基础研究

目的 探讨枸杞多糖（LBP-X）抑制人慢性粒细胞白血病（CML）K562细胞增殖的作用机制,研究LBP-X对K562细胞凋亡的影响,探讨LBP-X与三氧化二砷（As2O3）诱导K562细胞凋亡的剂量对应关系.方法 用LBP-X、As2O3处理K562细胞,四氮甲唑蓝（MTT）比色法检测药物对细胞的抑制作用,倒置显微镜观察细胞形态变化,流式细胞仪分析细胞DNA含量变化及凋亡细胞的比例.结果 LBP-X对K562细胞的IC50约为227.50 μg/ml,经LBP-X作用后,K562细胞呈现凋亡的形态学改变.流式细胞仪可在2倍体前检测到凋亡峰,凋亡细胞比例为8.43%-57.92%.结论 LBP-X对K562细胞有抑制作用,并能诱导其发生凋亡.LBP-X对K562细胞的效能和效价强度约为As2O3的1/1 000.     

Study on Taxol in Inhibiting Human Leukemia Cell Proliferation andInducing Apoptosis

Objective: To explore the effects of Taxol in inhibiting human leukemia k562 cell proliferation and inducing apoptosis in vitro. Methods: Human leukemia K562 cells were treated with Taxol of different concentrations for 12-72 hrs. Cell proliferation was evaluated by MTT assay and morphological changes of apoptosis were examined by microscopy. Cell apoptosis was determined by flow cytometry (FCM) and DNA gel electrophoresis. Results: Growth of K562 cells was inhibited by Taxol with an IC50 value of 0. 84μg/ml. Typical nuclear condensation and apoptosis bodies were observed as early as 24 hrs after a 0.5μg/ml Taxol treatment; Apoptotic rate of the Taxol-treated K562 cells increased from 3.7% to 24.0% in 24 hrs. No DNA ladder was observed by DNA gel electrophoresis. Conclusion: Taxol could inhibit K562 cell growth and induce apoptosis in vitro.     

SeO2诱导白血病细胞凋亡及对凋亡相关基因Bcl-2和p53表达调控的影响

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、凋亡及Bcl-2和p53表达水平的影响。方法 采用流式细胞术测定细胞的凋亡率及Bcl-2和p53的表达。结果 10和30mmol/LSeO2能抑制3种细胞增生。30mmol/LSeO₂作用48h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;能使NB4和K562细胞Bcl-2表达显著下降、促进p53的表达。结论 SeO₂对3种白血病细胞均有诱导凋亡作用,在凋亡过程中涉及了细胞内凋亡相关基因Bcl-2和p53表达和调控。