雷公藤甲素诱导肝细胞L-02凋亡的机制

目的:探讨雷公藤甲素对人正常肝细胞L-02细胞的凋亡诱导作用及细胞线粒体呼吸链复合体抑制剂对细胞凋亡的影响。方法:采用Annexin V-FITC/PI双染色法检测雷公藤甲素对L-02细胞的凋亡作用,JC-1染色观察细胞线粒体膜电位的变化,线粒体呼吸链复合体抑制剂对细胞活性氧释放及细胞凋亡的影响。结果:流式细胞仪检测结果显示,雷公藤甲素对细胞增殖的抑制作用与浓度、时间呈正相关;JC-1染色可见经雷公藤甲素作用的细胞线粒体膜电位降低;加入细胞线粒体呼吸链抑制剂后,与单用雷公藤甲素组比较,雷公藤甲素+rotenone组细胞活性氧释放水平显著降低(P<0.05),同时抑制剂合用组细胞凋亡百分率显著降低(P<0.05)。结论:雷公藤甲素体外可诱导L-02细胞凋亡,机制可能是与公藤甲素上调经线粒体呼吸链复合体Ⅰ、Ⅲ产生的活性氧有关。     

雷公藤甲素对人肝细胞L-02肝药酶活性的影响

目的:研究雷公藤甲素对正常人肝细胞株L-02细胞的损伤作用及其对肝CYP3A及CYP2E1蛋白表达量的影响。方法:体外培养L-02细胞,MTT法检测雷公藤甲素对L-02细胞的损伤,试剂盒测定培养基上清液中AST、ALT及AKP活性,Western-blot测定细胞中CYP3A及CYP2E1蛋白的含量。结果:雷公藤甲素对L-02细胞有损伤作用,且随雷公藤甲素浓度的增加或孵育时间的延长,其损伤作用增强,同时,培养基中AST、ALT及AKP的活性增强;雷公藤甲素诱导细胞中CYP3A蛋白表达减弱,CYP2E1蛋白表达增强。结论:雷公藤甲素体外对人正常肝细胞L-02细胞有损伤作用,且能影响肝CYP450酶系的表达。     

雷公藤甲素对Caspase诱导人肝细胞L-02凋亡机制的研究

目的：研究雷公藤甲素诱导正常人肝细胞株L-02细胞凋亡的机制。方法：体外培养L-02细胞。MTT法检测雷公藤甲素的细胞毒性作用;Annexin V-FITC/PI双染法检测雷公藤甲素对L-02细胞的凋亡诱导作用;测定细胞基质中半胱天冬氨酸酶（Caspase）8、Caspase9与Caspase3活性的变化及加入Caspase抑制剂Z-DEVD-FMK后对细胞凋亡的影响。结果：雷公藤甲素能显著诱导L-02细胞的凋亡,其作用呈明显的量效关系与时间依赖性;120nmol·L-1雷公藤甲素作用L-02细胞24h使Caspase3的活性显著增强;加入Z-DEVD-FMK后,能有效抑制雷公藤甲素诱导的细胞凋亡。结论：雷公藤甲素体外诱导正常人肝细胞株L-02细胞凋亡可能与上调Caspase3、9的活性有关。     

雷公藤甲素诱导人肝细胞凋亡的机制

目的 探讨雷公藤甲素诱导人肝细胞L-02凋亡的机制.方法 将L-02细胞分为对照组和雷公藤甲素50 nM实验组,处理48 h后,采用MTT法检测细胞存活率,流式细胞术检测细胞凋亡率、活性氧(ROS)的荧光强度,试剂盒检测细胞色素C的浓度以及半胱天冬氨酸蛋白酶9(Caspase-9)和Caspase-3的活性,Western blot检测细胞中B细胞淋巴瘤-白血病2(Bcl-2)和Bcl相关X蛋白(Bax)的表达.结果 与对照组相比,实验组细胞存活率下降,细胞凋亡率上升,Bcl-2表达下调,Bax表达、细胞色素C浓度、ROS荧光强度、Caspase-9和Caspase-3活性均明显上调(P＜0.05或P＜0.01).结论 雷公藤甲素可以抑制L-02细胞的存活,通过激活线粒体凋亡途径促进L-02细胞凋亡,下调Bcl-2与Bax比率,促进细胞色素C释放,诱导ROS生成,进一步破坏线粒体膜,增加细胞色素C的释放,激活Caspase-9和Caspase-3介导的细胞凋亡通路,最终诱导细胞凋亡.     

基于Nrf2信号通路探讨异甘草酸镁对雷公藤甲素损伤L-02细胞中相关蛋白表达的影响

目的基于Nrf2-ARE信号通路探讨异甘草酸镁对雷公藤甲素损伤L-02细胞的保护作用及相关机制。方法体外培养正常人肝L-02细胞,通过免疫荧光技术检测异甘草酸镁(60μmol·L^-1)对雷公藤甲素(80nmol·L^-1)损伤L-02细胞中Nrf2核转移的影响;采用Western blotting测定细胞核中Nrf2及细胞中细胞色素P450 3A4(CYP3A4)、多药耐药相关蛋白2(multidrug resistance associated protein 2,MRP2)和胆盐输出泵(bile salt export pump,BSEP)的蛋白表达。结果雷公藤甲素对L-02细胞核中Nrf2具有诱导作用(P<0.01),并促进Nrf2核内转移;异甘草酸镁预处理组较雷公藤甲素组降低L-02细胞核中Nrf2的表达(P<0.01);异甘草酸镁预处理后对雷公藤甲素损伤L-02细胞中CYP3A4、MRP2和BSEP蛋白表达具有诱导作用(P<0.01)。结论异甘草酸镁可减轻雷公藤甲素对L-02细胞造成的损伤作用,其机制可能与诱导细胞核中Nrf2以及细胞中CYP3A4、MRP2和BSEP受体有关。     

雷公藤甲素对人肺腺癌A549细胞增殖和凋亡的影响

目的：研究雷公藤甲素对人肺腺癌‰细胞增殖和凋亡的影响。方法：采用MTT实验检测雷公藤甲素对体外培养的人肺腺癌A谢细胞增殖的影响，运用流式细胞术、基因组DNA电泳观察凋亡特征性“梯状”条带检测细胞凋亡，采用caspase活性定量检测试剂盒分析caspase-9，3的活化。结果：雷公藤甲素对体外培养的A谢细胞具有明显的抑制作用。流式细胞仪检测显示雷公藤甲素可以显著诱导‰细胞凋亡，DNA电泳显示雷公藤甲素作用于A谢细胞后出现凋亡细胞特有的DNA阶梯状条带。雷公藤甲素作用后‰细胞caspase-9，caspase-3活性明显升高。结论：雷公藤甲素可能通过线粒体信号通路抑制肺腺癌A谢细胞的生长并诱导其凋亡。     

L-02肝细胞蛋白质双向电泳条件的建立

人类蛋白质组学的研究是揭示人类生命活动和疾病机制的最终阶段,运用蛋白质组学技术寻找各种疾病的关键蛋白和标志蛋白,可实现更加及时、准确的诊断[1]。双向电泳技术一个主要的应用领域是“蛋白质组分析”,包括对来自一个样品的大量蛋白质同时进行系统地分离、识别和定量。本实验通过比较几种不同的蛋白处理方法,优化双向电泳条件,找出较佳的样本处理方式,为成功进行双向电泳及质谱鉴定提供基础。1材料与方法1·1细胞株L-02肝细胞购自中国科学院上海细胞生物研究所。1·2试剂与仪器二羟乙基呱嗪乙烷磺酸(HEPES)、噻唑蓝均为美国Sigma产…     

硒对六价铬诱导L-02肝细胞DNA损伤的影响

目的研究硒(Se)对六价铬Cr(Ⅵ)诱导肝细胞DNA损伤的影响,探讨Se对Cr(Ⅵ)中毒预防的可能性。方法分别用8和32μmol/L重铬酸钾(K2Cr2O7)溶液处理在含有0.1和10μmol/L亚硒酸钠(Na2SeO3)中培养的L-O2细胞,采用单细胞凝胶电泳(SCGE)检测L-02肝细胞DNA损伤程度。结果8和32μmol/L K2Cr2O7单独作用时,可引起L-02肝细胞DNA损伤。当Na2SeO3与K2Cr2O7联合作用时,0.1μmol/L NaSeO3对8、32μmol/L K2Cr2O7诱导的DNA损伤存在明显的拮抗作用,而10μmol/L Na2SeO3单独存在与K2Cr2O7相互作用均具有诱导DNA损伤的作用。结论Se对L-02肝细胞DNA损伤的影响具有剂量依赖性,即Se在较低处理组水平具有降低Cr(Ⅵ)对L-02肝细胞DNA损伤的作用,在高处理组水平能诱导细胞DNA损伤。     

雷公藤醇提物对人L02肝细胞增殖和凋亡的作用

目的 研究雷公藤醇提物对人L02肝细胞增殖和凋亡的作用.方法 以0、4、8、16 mg/L的雷公藤醇提物(分别为对照组、4 mg/L组、8 mg/L组、16 mg/L组)干预体外培养的人L02肝细胞48 h后,采用四甲基偶氮唑蓝(MTT)法检测各组肝细胞的生长抑制率,采用膜联蛋白V-异硫氰酸荧光素/碘化吡啶双染流式细胞术检测人L02肝细胞的凋亡率.结果 与对照组比较,4mg/L组的吸光度值降低[(0.90±0.04)比(0.94±0.06),P＜0.05];当雷公藤醇提物浓度为8 mg/L和16 mg/L时,细胞液吸光度值降低均更加明显[(0.76±0.05)、(0.53 ±0.04)比(0.94±0.06),均P＜0.01].与对照组比较,4 mg/L组的细胞凋亡率升高,差异有统计学意义[(9.68±0.45)％比(5.42±0.27)％,P＜0.05];当雷公藤醇提物浓度为8mg/L和16 mg/L时,人L02肝细胞凋亡率升高均更加明显[(30.19±0.31)％、(57.26±0.54)％比(5.42±0.27.)％,均P＜0.01].结论 雷公藤醇提物对人L02肝细胞的增殖有抑制作用,并可诱导其凋亡.     

纳米锰锌铁氧体对肝细胞L-02的细胞毒性

目的 研究纳米锰锌铁氧体对正常人肝细胞株(L-02)的体外毒性.方法 使用透射电镜(TEM)以及X射线衍射仪(XRD)对纳米锰锌铁氧体进行表征,采用噻唑蓝实验(MTT比色法)检测M<,0.5>2Zn<,0.5>Fe<,2>O<,4>纳米材料不同浓度(6.25、25、50、100、200、400和800μg/ml)以及不...     

Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells

Aim:

To investigate the role of reactive oxygen species (ROS) in oridonin-induced apoptosis and autophagy in HeLa cells.

Methods:

The cell viability was measured using MTT assay. Morphological changes of apoptosis and autophagy were examined using Hoechst 33258 staining and monodansylcadaverine (MDC) staining, respectively. The mitochondrial membrane potential (ΔΨm) was measured using fluorescent dye rhodamine 123. DCF-induced fluorescence was used to measure the intracellular ROS level. Protein expression was examined using Western blot.

Results:

Treatment of HeLa cells with oridonin (20–160 μmol/L) inhibited the cell growth in time- and concentration-dependent manners. The cells treated with oridonin (80 μmol/L) for 24 h displayed marked DNA fragmentation and MDC-positive autophagosomes. In the presence of the specific autophagy inhibitor 3-MA (2 mmol/L), the oridonin-induced apoptosis was significantly enhanced. Treatment of HeLa cells with oridonin (20–120 μmol/L) induced intracellular ROS generation in a concentration-dependent manner. In the presence of the ROS scavenger NAC (5 mmol/L), the oridinin-induced ROS generation was markedly reduced. NAC (5 mmol/L) or non-thiol antioxidant catalase (1000 U/mL) significantly reduced the oridonin-induced inhibition of cell growth and apoptosis. Furthermore, oridonin significantly reduced ΔΨm, which was blocked by NAC. Oridonin markedly increased Bax expression in mitochondria, and decreased Bcl-2 expression in both the cytosol and mitochondria. Oridonin also markedly increased the phosphorylation of Bcl-2 in the cytosol. All the effects were blocked by NAC. Oridonin increased the levels of caspase-3 and caspase-8, and decreased the expression of pro-caspase 3 and pro-caspase 9, which were blocked by NAC.

Conclusion:

ROS plays a critical role in oridonin-induced apoptosis and autophagy.     

活性氧类物质在化学物诱导的细胞凋亡中的调节作用(英文)

细胞凋亡是在生理或病理条件下 ,为维持内环境的稳定的一种程序性细胞死亡。诱导细胞凋亡的因素可分为物理性、化学性和生物性因素。很多实验数据表明氧化应激在细胞凋亡的发生过程中具有重要作用。各种活性氧类物质如超氧阴离子、过氧化氢、羟自由基和一氧化氮均与细胞凋亡的发生有关。但是活性氧类物质诱导凋亡发生的机理尚未阐明。本综述在对细胞凋亡和氧化应激描述的基础上着重讨论活性氧类物质诱导细胞凋亡发生的可能机理。在以后的研究中 ,将着重于探讨化学物质通过产生活性氧类物质而影响细胞凋亡的机理及建立相关的生物标志物。     

Induction of apoptosis and reactive oxygen species production by N-nitrosopiperidine and N-nitrosodibutylamine in human leukemia cells

N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) belong to a group of N-nitrosamines that are widely distributed in foodstuffs and the occupational environment. In the present study, the human promyelocytic leukemia cell line HL-60, was used to characterize the apoptotic effects of N-nitrosamines, and to examine the production of reactive oxygen species (ROS). Apoptotic cells were identified by (i) chromatin condensation (ii) flow cytometry analysis and (iii) poly(ADP-ribose) polymerase (PARP) cleavage. NPIP and NDBA induced morphological changes consistent with apoptotic events in HL-60 cells. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-manner. It was observed that NDBA was stronger than NPIP, since it induced a significant apoptotic cell death after 18 h starting from a concentration of 2 mm, whereas NPIP was effective at 10 mm. Furthermore, PARP was markedly cleaved with 0.5 mm of NDBA and 5 mm of NPIP after treatments for 3 and 18 h, respectively. Finally, the ROS level was found to be elevated after 0.5 h of treatment with both N-nitrosamines. Antioxidant N-acetylcysteine (NAC) completely inhibited the ROS production induced by NPIP and NDBA. However, this action seems not to be associated with the apoptosis because NAC did not block N-nitrosamines-induced apoptosis. The data demonstrate that NPIP and NDBA induce apoptosis and ROS production in HL-60 cells.     

白藜芦醇诱导K562细胞凋亡过程中活性氧水平的改变

目的：探讨白藜芦醇对K562细胞的凋亡诱导效应和对K562细胞内活性氧水平的影响。方法：应用噻唑蓝(MTr)比色法、光镜、DNA琼脂糖凝胶电泳和流式细胞术(FCM)检测细胞凋亡；FCM测定细胞内活性氧(ROS)水平。结果：白藜芦醇显著抑制K562细胞增殖，并呈现典型的凋亡形态学改变；DNA电泳可见梯状条带出现；FCM分析显示S期细胞数明显增多，出现S／G，期阻滞，ROS水平升高。结论：白藜芦醇诱导K562细胞凋亡与细胞内活性氧水平升高有关。     

Quinalizarin induces cycle arrest and apoptosis via reactive oxygen species-mediated signaling pathways in human melanoma A375 cells

Quinalizarin, a bioactive and highly selective compound, is known to promote apoptosis in colon and lung cancer cells. However, studies evaluating quinalizarin-induced apoptosis in melanoma cells have not been conducted. In the present study, we investigated the underlying mechanisms of antimelanoma activity of quinalizarin in human melanoma A375 cells. The MTT assay and Trypan blue staining were used to evaluate the cell viability. The flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Western blot was used to detect the expression of cell cycle and apoptosis-related proteins, MAPK, and STAT3. The results revealed a significant dose and time dependent effect of quinalizarin on inhibiting proliferation in three kinds of human melanoma cells, and had no significant toxic effects on normal cells. Moreover, quinalizarin triggered G2/M phase cell arrest by modulating the protein expression levels of CDK 1/2, cyclin A, cyclin B, p21 and p27, and induced apoptosis by down-regulating the antiapoptotic protein Bcl-2 and upregulating the proapoptotic protein BAD, leading to the activation of caspase-3 and PARP in the caspase cascade in A375 cells. Quinalizarin treatment led to apoptosis of A375 cells via activation of MAPK and inhibition of STAT3 signaling pathways. In addition, quinalizarin increased the level of ROS, but ROS scavenger NAC inhibited quinalizarin-induced apoptosis by regulating MAPK and STAT3 signaling pathways. In summary, quinalizarin induces cell cycle arrest and apoptosis via ROS-mediated MAPK and STAT3 signaling pathways in human melanoma A375 cells, and quinalizarin may be used as a novel and effective antimelanoma therapeutic.     

辛伐他汀诱导K562细胞凋亡及对细胞内活性氧和Ca2+动态变化的影响

目的研究辛伐他汀诱导人红白血病细胞株K562细胞凋亡及细胞内活性氧与Ca2+水平的变化,以探讨凋亡机制.方法20 μmol·L-1辛伐他汀处理K562细胞,24 h 后光镜观察细胞形态;流式细胞术检测细胞凋亡率、活性氧和细胞内游离Ca2+水平.结果20 μmol·L-1辛伐他汀作用K562细胞 48 h 后出现核固缩、核碎裂和凋亡小体等形态学改变;AnnexinV-FITC/PI检测细胞早期凋亡率,处理组凋亡率高于对照组,随药物作用时间延长逐渐增大,具有时间依赖性.荧光染料2',7'-二氯荧光乙酰乙酸(2',7'-dichloro fluorescein diacetate, DCFH-DA)检测K562细胞内活性氧,不同时间处理组与对照组比较活性氧均升高,峰值时间为 24 h,与对照组比较发生显著改变.荧光染料Fluo-3AM检测K562细胞内游离Ca2+浓度,不同时间细胞内游离Ca2+浓度均升高,峰值时间为 12 h,与对照组比较发生显著变化.结论辛伐他汀诱导K562细胞凋亡的可能机制是通过提高细胞内活性氧及游离Ca2+水平,从而导致细胞凋亡.     

Requirement of reactive oxygen species generation in apoptosis of leukemia cells induced by 2-methoxyestradiol

Aim： To investigate the effects of 2-methoxyestradiol （2-ME） on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms. Methods： Human myeloid leukemia cells HL-60 and U937 were used. Measurement ofmitochondrial membrane potential （Dym） was performed using 5,5′＇,6,6′-Tetrachloro-1, 1′,3,3′- tetraethylbenzimidazolylcarbocyanine iodide （ JC- 1）. Apoptosis and cellular nitric oxide （NO） were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium （DHE）. Cytotoxicity was analyzed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl- tetrazolium assay. Results： 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species （ROS）, including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME. Conclusion： Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.     

Multiwall carbon nano‐onions induce DNA damage and apoptosis in human umbilical vein endothelial cells

Growing evidence has indicated the potential adverse effects on cardiovascular system of some nanomaterials, including fullerenes. In this study, we have evaluated the biological effects of multiwall carbon nano‐onions (MWCNOs) (average size of 31.2 nm, ζ potential of 1.6 mV) on human umbilical vein endothelial cells (HUVECs). It was found that MWCNOs exhibited a dose‐dependent inhibitory effect on cell growth; EC₅₀ was 44.12 μg/mL. Thus, three concentrations were chosen (0.2, 1, and 5 μg/mL) for further experiments. Flow cytometry analysis revealed that 1 and 5 μg/mL MWCNOs could induce apoptosis in HUVECs, the apoptotic rates were 12% and 24% at 24 h after exposure. On the other hand, MWCNOs did not affect the cell cycle distribution during 24 h period. Using γH2AX foci formation as an indicator for DNA damage, it was shown that 5 μg/mL MWCNOs can induce γH2AX foci formation in HUVECs at 6, 12, and 24 h after treatment, whereas 0.2 μg/mL MWCNOs induced γH2AX foci formation only at 6 h after treatment. In addition, all three concentrations of MWCNOs induced the generation of reactive oxygen species (ROS), and inhibition of ROS generation can partially decrease the γH2AX foci formation induced by MWCNOs. Taken together, these data first suggested that MWCNOs can induce DNA damage and apoptosis in HUVECs, and that ROS might be involved in this process. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 442–450, 2013.     

雷公藤甲素诱导大鼠主动脉平滑肌细胞凋亡的实验研究

目的　观察雷公藤甲素 (T10 )对平滑肌细胞 (SMCs)细胞周期进程的影响和诱导凋亡作用。方法　体外主动脉SMCs培养和流式细胞仪技术。结果　T10 与指数增殖期SMCs共育后 ,可引起核染色质浓缩、聚集 ,细胞膜内陷 ,凋亡小体形成等凋亡形态学改变。流式细胞仪分析 ,在G1峰前出现凋亡峰 ,峰值随T10 浓度增加而升高 ;G1期细胞进入S期并阻滞于S期 ;G1、M期细胞比例下降而S期升高 ;凋亡分子Fas表达明显升高。结论　T10 可以诱导指数增殖期SMCs凋亡