三氧化二砷诱导乳腺癌细胞株MCF-7凋亡对端粒酶活性的影响

<正>乳腺癌是严重威胁妇女健康的恶性肿瘤之一,近年发病率逐年上升,且发病年龄也趋于年轻化。近年来研究发现As2O3不仅对APL细胞,而且对其他血液肿瘤细胞和许多实体瘤细胞均具有诱导凋亡作用。Wang等研究表明端粒酶与恶性肿瘤发生有着紧密的关联,p21特异性核苷     

太白楤木皂苷对砷诱导的肝细胞线粒体途径凋亡的保护作用

目的探讨太白楤木皂苷对砷暴露损伤的保护作用及分子机制。方法人正常肝细胞HL-7702进行砷暴露及太白楤木皂苷保护处理。砷暴露组用10μmol L-1终浓度的NaAsO2处理,太白楤木皂苷保护组用不同浓度太白楤木皂苷预处理12 h后,再进行砷暴露处理。24 h后MTT法检测细胞活力;12 h后收集细胞样品,分别检测细胞的凋亡、活性氧（ROS）、还原型谷胱甘肽（GSH）,氧化损伤产物丙二醛（MDA）水平以及线粒体凋亡途径的重要因子细胞色素C（Cyto C）释放和Caspase-3的激活情况。结果砷暴露可以引起肝细胞活力显著降低,给予不同浓度的太白楤木皂苷具有一定的保护作用,其中5μg mL-1太白楤木皂苷保护效果最佳。砷暴露可以引起细胞凋亡比例显著增加,细胞ROS水平升高,GSH水平降低,氧化损伤产物MDA水平增加,线粒体凋亡途径因子Cyto C和Caspase-3的激活。太白楤木皂苷（5μg mL-1）预处理能够显著拮抗砷暴露引起的细胞上述指标变化。结论砷暴露可以通过诱导细胞氧化应激,激活线粒体途径细胞凋亡,发挥其损伤毒性作用。太白楤木皂苷可以通过抑制细胞氧化应激,抑制线粒体途径的细胞凋亡,从而发挥其抗砷暴露损伤的保护作用。     

三氧化二砷诱导胃癌细胞SGC-7901凋亡及对NF-κB表达的影响

目的:观察三氧化二砷(As2O3)诱导胃癌细胞SGC-7901凋亡及对NF-κB表达的影响。方法:流式细胞仪(FCM)检测不同浓度As2O3作用后48 hSGC-7901的凋亡率,凝胶电泳迁移改变分析法(EMSA)及免疫组织化学法检测As2O3处理48 h后NF-κB活性及表达。结果:As2O3可诱导SGC-7901凋亡(P<0.05)并降低NF-kB的活性及表达(P<0.05),有浓度依赖性。结论:As2O3能诱导SGC-7901凋亡,并降低NF-κB活性及表达,这可能是As2O3抗肿瘤的机制之一。     

人肾小管上皮细胞及正常肝细胞在药物毒性器官选择中的应用

目的：探讨人肾小管上皮细胞（HK－2）及人正常肝细胞（L－02）在药物毒性器官选择中的应用。方法以肾毒性药物庆大霉素、顺铂、环孢素A及肝毒性药物硫唑嘌呤作用于HK－2及L－02细胞24 h,考察并比较药物对2种细胞的形态、存活、凋亡及酶学指标变化的影响。结果肾毒性药物在较低浓度下即可引起HK－2细胞的形态、存活、凋亡及酶学指标的显著性变化,肝毒性药物则在较低浓度下即可引起L－02细胞相关指标的显著性变化;比较4种药物的IC10值发现：3种肾毒性药物对HK－2的IC10值均小于对L－02的IC10值;而肝毒性药物硫唑嘌呤的IC10值则HK－2大于L－02。结论基于HK－2及L－02细胞评价药物的毒性器官选择性是可行性的。     

三氧化二砷对人胶质瘤细胞株U251影响的实验研究

目的 探讨三氧化二砷(As2O3)抗人胶质瘤细胞株U251的作用及机制。方法采用0．5、1．0、2．0、4．0μmol／L AS2O3诱导人胶质瘤细胞株U251,于培养第1、2、3、4、5、6d应用倒置相差显微镜、四甲基偶氮唑蓝法、流式细胞仪观察人胶质瘤细胞株U251的存活,形态学改变及细胞DNA含量的变化。结果0．5～4μmol／L的As2O3明显抑制U251的增殖。流式细胞仪分析显示,加药组在G1期细胞前均出现亚二倍体峰,且G0/G1期细胞减少,诱导细胞凋亡。结论体外三氧化二砷能抑制人胶质瘤细胞U251增殖,提示有希望用于神经胶质瘤患者的治疗。     

缬沙坦对人肾近端小管上皮细胞TGF-β/Smad信号途径的影响

目的观察缬沙坦对TGF-β刺激人肾近端小管上皮细胞表达Smad的影响。方法以人肾小管上皮细胞(HKC)为对象,分为正常对照组;TGF-β1刺激组;缬沙坦组。Western blot检测p-Smad2/3、smad2/3、Smad7以及细胞上清ColⅠ蛋白含量;RT-PCR检测Smad2 mRNA和Smad7 mRNA水平。MTT法检测刺激组及缬沙坦组在不同时间、不同药物浓度对细胞增殖状态的影响。结果①Western blot显示,与TGF-β1刺激组相比,缬沙坦使Smad2/3、p-Smad2/3蛋白的表达下降,ColⅠ分泌显示相同趋势,而Smad7蛋白的表达没有变化。②半定量RT-PCR显示,与TGF-β1刺激组相比,缬沙坦组Smad2 mRNA降低,Smad7 mRNA无变化。③MTT法检测显示不同浓度缬沙坦对TGF-β1刺激所引起的细胞增殖受抑制现象均有改善,并且随药物浓度的增加该作用增强。结论提示缬沙坦的肾脏保护作用可能部分是通过抑制TGF-β/Smads信号途径实现的。     

Mn2+对庆大霉素导致的人肾小管上皮细胞系损伤的保护作用

目的观察Mn2 对庆大霉素导致的人肾小管上皮细胞系的损伤有保护作用,并探讨其机制。方法用庆大霉素作用于人肾小管上皮细胞系(HK-2),造成肾损伤模型,MTT法检测Mn2 对细胞增殖活力的影响;分光光度法检测Mn2 导致的SOD、LDH活力和NAG酶含量的改变;电镜观察Mn2 作用后HK-2细胞微结构改变。结果Mn2 可使庆大霉素损伤的HK-2细胞增殖活力增加,降低LDH酶活力,减少NAG酶含量,升高SOD酶活性,减轻细胞线粒体肿胀程度和减少溶酶体膜的破裂。结论Mn2 对庆大霉素导致的人肾小管上皮细胞系的损伤有保护作用。其机制可能与减轻细胞氧化损伤、降低线粒体肿胀程度、保护溶酶体的完整性和减少溶酶漏出有关。     

三氧化二砷通过JNK信号通路诱导K562细胞凋亡

目的:探讨c-Jun氨基末端激酶(JNK)信号通路在三氧化二砷(As2O3)诱导K562细胞凋亡中的作用及机制。方法:体外培养K562细胞,用As2O3及特异性JNK抑制剂SP600125对K562细胞进行处理;倒置相差显微镜下观察细胞形态学变化;MTT法检测不同时间点细胞增殖抑制率;AnnexinV/PI染色结合流式细胞术检测细胞凋亡率;ELISA检测p-JNK蛋白表达的变化;流式细胞术检测突变型P53表达。结果:ELISA显示4μmol/LAs2O3作用48h后p-JNK蛋白表达增强,经SP600125预处理后,As2O3诱导的K562细胞p-JNK蛋白表达明显减弱(P<0.01),As2O3诱导的细胞增殖抑制率和细胞凋亡率均下降,与As2O3单作用组相比突变型P53表达增加(P<0.05)。结论:JNK信号转导通路在As2O3诱导K562细胞凋亡过程中发挥重要作用,是As2O3诱导K562细胞凋亡的主要途径之一。     

As_2O_3诱导人肝癌细胞凋亡及对Fas表达和钙含量的影响

化疗在原发性肝癌的综合治疗中占重要地位.为寻找肝癌治疗新的有效药物,我们观察了As2O3对人肝癌细胞株BEL7402的生物学效应,以期为As2O3临床治疗肝癌提供实验依据.     

Endoplasmic reticulum stress contributes to arsenic trioxide‐induced intrinsic apoptosis in human umbilical and bone marrow mesenchymal stem cells

Arsenic trioxide is an old drug and has been used for a long time in traditional Chinese and Western medicines. However, the cancer treatment of arsenic trioxide has heart and vascular toxicity. The cytotoxic effects of arsenic trioxide and its molecular mechanism in human umbilical mesenchymal stem cells (HUMSC) and human bone marrow‐derived mesenchymal stem cells (HMSC‐bm) were investigated in this study. Our results showed that arsenic trioxide significantly reduced the viability of HUMSC and HMSC‐bm in a concentration‐ and time‐dependent manner. Arsenic trioxide is able to induce apoptotic cell death in HUMSC and HMSC‐bm, as shown from the results of morphological examination, flow cytometric analyses, DAPI staining and comet assay. The appearance of arsenic trioxide also led to an increase of intracellular free calcium (Ca²⁺) concentration and the disruption of mitochondrial membrane potential (ΔΨm). The caspase‐9 and caspase‐3 activities were time‐dependently increased in arsenic trioxide‐treated HUMSC and HMSC‐bm. In addition, the proteomic analysis and DNA microarray were carried out to investigate the expression level changes of genes and proteins affected by arsenic trioxide treatment in HUMSC. Our results suggest that arsenic trioxide induces a prompt induction of ER stress and mitochondria‐modulated apoptosis in HUMSC and HMSC‐bm. A framework was proposed for the effect of arsenic trioxide cytotoxicity by targeting ER stress. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 314–328, 2016.     

庆大霉素对人肾小管上皮细胞系(HK-2)热休克蛋白(HSP)70表达的影响

目的观察了庆大霉素在体外对肾小管上皮细胞内HSP70表达的影响。方法将庆大霉素(100mg·L-1)作用于人肾小管上皮细胞系(HK-2),用MTT法检测庆大霉素对HK-2细胞增殖活力的影响,应用免疫细胞化学和Westernblot法观察了HSP70蛋白表达的改变,采用RT-PCR法检测HSP70mRNA表达的变化。结果庆大霉素在作用HK-2细胞24、48、72h后,MTT吸光度值为0·166±0·02、0·181±0·009、0·237±0·016,均低于对照组0·187±0·011、0·32±0·016、0·515±0·035(P<0·01);HSP70蛋白阳性表达率(%)分别为69±17、86±21、89±25,均高于对照组6±3、4±3、7±2(P<0·01);还可增加HSP70蛋白和mRNA表达(P<0·01)。结论庆大霉素在降低人肾小管上皮细胞系细胞增殖活力的同时,增加HSP70的表达。     

Morphologic and functional alterations induced by low doses of mercuric chloride in the kidney OK cell line: ultrastructural evidence for an apoptotic mechanism of damage

Mercury produces acute renal failure in experimental animal models, but the mechanism of tubular injury has not completely been clarified. There is an increased interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. However, detailed studies of morpho-functional alterations induced by mercuric chloride in kidney cell lines are scarce. This work characterizes these alterations in OK cell cultures. Morphological alterations were profiled using light microscopy, transmission electron microscopy, and confocal microscopy, as well as mitochondrial functional assays in the cells exposed to low concentrations of HgCl₂. At concentrations of 1 and 10 μM of HgCl₂ there were no morphological or ultrastructural alterations, but the mitochondrial function (MTT assay) and intracellular ATP content was increased, especially at longer incubation times (6 and 9 h). At 15 μM HgCl₂, both the mitochondrial activity and the endogenous ATP decreased significantly. At this concentration the OK cells rounded up, had increased number of cytoplasmic vacuoles, and detached from the cell monolayer. At 15 μM HgCl₂ ultrastructural changes were characterized by dispersion of the ribosomes, dilatation of the cisterns of the rough endoplasmic reticulum, increase of number of cytoplasmic vacuoles, chromatin condensation, invaginations of the nuclear envelope, presence of cytoplasmic inclusion bodies, and alterations in the size and morphology of mitochondria. At 15 μM HgCl₂ apoptotic signs included membrane blebbing, chromatin condensation, mitochondrial alterations, apoptotic bodies, and nuclear envelope rupture. Using confocal microscopy and the mitochondrial specific dye MitoTracker Red, it was possible to establish qualitative changes induced by mercury on the mitochondrial membrane potential after incubation of the cells for 6 and 9 h with 15 μM HgCl₂. This effect was not observed at short times (1 and 3 h) with this same concentration, neither with 1 and 10 μM HgCl₂ in all the studied times. Taken together, these findings indicate that low concentrations of HgCl₂ induce apoptosis by inhibiting mitochondrial function, and the OK cell line may be considered a useful tool for the study of programmed cell death involving mercurial species and other heavy metals.     

人肝癌细胞和肝细胞放射敏感性的体外研究

目的 观察人肝癌细胞株HepG2及人肝细胞株QSG-7701对高能X线照射的敏感性和时相差异.方法 用吸收剂量为5 Gy的6 MV X线单次照射体外培养的HepG2细胞及QSG-7701细胞,以MTT比色法测定细胞在受照后不同时间的存活率,分别采用流式细胞术和激光共聚焦显微镜观测细胞凋亡率和形态变化.结果 高能X线照射后6h HepG2细胞凋亡率达到高峰(32%),存活率最低(62%),而QSG-7701细胞在照射后24h凋亡率和存活率才分别达高峰(17%)和低谷(82%).两株细胞单次照射后6h的凋亡率和存活率差异最大.结论 高能X线能够诱导人肝癌细胞株HepG2及人肝细胞株QSG-7701发生凋亡,两株细胞存在不同的敏感性和时相差异,HepG2细胞对射线敏感性比QSG-7701细胞高,HepG2细胞凋亡高峰明显早于QSG-7701细胞.     

白英提取物诱导人肝癌BEL—7404细胞凋亡作用

探讨中药白英提取物对肝癌BEL－7404细胞系的凋亡作用。方法 用不同浓度的白英提取物处理肝癌BEL－7404细胞,通过观察细胞形态、DNA凝胶电泳法研究白英提取物诱导肝癌BEL－7404细胞产生凋亡现象。结果 光学显微镜下可见细胞呈凋亡特征性变化,凝胶电泳可见细胞DNA有规律断裂形成梯形图谱。结论 白英可诱导肝癌BEL－7404细胞产生凋亡。     

万古霉素诱导人肾小管上皮细胞损伤及其Smad4表达的研究

目的 研究万古霉素诱导人肾小管上皮细胞（HK-2）损伤及其Smad同源物4（Smad4）表达，并探讨其潜在的作用机制。方法 使用不同终浓度（0、1、2、3、4、5 mmol/L）的万古霉素溶液作用于HK-2细胞24 h，显微镜下观察万古霉素对细胞形态的影响；蛋白质免疫印迹法检测Smad4、B淋巴细胞瘤-2（Bcl-2）相关X蛋白（Bax）、Bcl-2的蛋白水平；TUNEL法检测HK-2细胞凋亡情况；CCK8法检测HK-2细胞活力来研究万古霉素对肾脏的毒性作用。构建Smad4过表达质粒，通过CCK8法和TUNEL实验检测Smad4过表达对万古霉素诱导的HK-2细胞损伤的影响。结果 与对照组比较，随着万古霉素浓度升高，HK-2细胞逐渐变形，数量逐渐减少；细胞活力显著降低，凋亡率显著增加（P＜0.05、0.01）。与对照组比较，促凋亡蛋白Bax表达显著增高，而抗凋亡蛋白Smad4、Bcl-2表达则显著降低（P＜0.05、0.01、0.001）。过表达Smad4后，使万古霉素诱导的HK-2的细胞活力显著增加，凋亡率显著降低（P＜0.05、0.001）。结论 万古霉素能够诱导人肾小管上皮细胞损伤，其作用机制可能与调控Smad4的蛋白降解密切相关。     

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

四种砷拮抗剂对三氧化二砷诱导的三种粒系白血病细胞凋亡和端粒酶活性的减量调节作用(英文)

目的:研究巯基供体N-乙酰半胱氨酸(NAC)和二巯丁二钠(NDMS)、抗氧化剂过氧化氢酶(CAT)和Ca~(2 )清除刘(Quin 2)对三氧化二砷诱导的三种粒系白血病细胞凋亡和端粒酶活性改变的调控作用。方法:用流式细胞仪和PCR ELISA法分别检测NAC、NDMS、CAT或Quin 2与三氧化二砷共同作用于三种粒系白血病细胞后其凋亡和端粒酶活性的变 化。结果:三氧化二砷0.6、2.7和8.1μmol/L可分别诱导急性早幼粒细胞白血病细胞株NB4,慢性粒细胞白血病细胞株K562,急性粒细胞白血病细胞株HL-60细胞发生40％-60％的凋亡,同时下调三种细胞的端粒酶活性。NAC 4mmol/L,NDMS 200μmol/L,CAT 80kU/L,Quin 220μmol/L不同程度抑制这种凋亡作用。NAC和CAT既可独立降低三种细胞的端粒酶活性,也可促进三氧化二砷对端粒酶的下调作用,而Quin 2可抑制K562和HL-60细胞中的这种下调作用。结论:三氧化二砷诱导的三种细胞的凋亡过程涉及了疏基失活、自由基的改变、细胞内Ca~(2 )浓度改变及端粒酶活性下降。NAC、NDMS、CAT及Quin 2可不同程度拮抗三氧化二砷对三种细胞的作用。     

Dose-dependent induction of apoptosis by R-apomorphine in CHO-K1 cell line in culture

A variety of mechanisms have been proposed as explanations for the distinctive neuropathology of Parkinson's disease, such as increased iron levels, increased oxidant stress or decreased antioxidant defences. The vulnerability of dopamine-containing neurons towards cell death has attracted much attention to the dopamine molecule itself as one of the probable neurotoxic factors leading to neurodegeneration. The similarity between apomorphine and dopamine with regards to their chemical, pharmacological and toxicological properties provided a basis for investigating the nature of the toxicity of the former agent. In this study the CHO-K1 cell line was exposed to different concentrations of apomorphine, and markers of cell death and apoptosis were studied. Apomorphine reduced cell proliferation in a dose-dependent fashion after 72 h incubation. Furthermore, apomorphine induced dose-dependent cell death at concentrations of 10-50 microM. The CHO-K1 line showed specific markers of apoptosis such as the typical DNA laddering phenomenon on agarose gel, morphological changes of apoptotic nuclei as described by in situ end labelling, and annexin binding.These data strongly suggest that apomorphine, like dopamine, elicits its cytotoxic effect with an apoptotic mechanism.     

左炔诺孕酮诱导体外人子宫肌瘤细胞凋亡的抗肿瘤机制初步研究

目的：探讨左炔诺孕酮诱导体外人子宫肌瘤细胞凋亡的抗肿瘤机制。方法：单层细胞培养后,使用MTT法分析左炔诺孕酮对人子宫肌瘤细胞增殖作用;使用流式细胞术分析左炔诺孕酮对人子宫肌瘤细胞周期与凋亡的影响;使用Western-blot法分析左炔诺孕酮对人子宫肌瘤细胞P53凋亡通路的影响。结果：左炔诺孕酮对人子宫肌瘤细胞的增殖具有明显抑制作用;左炔诺孕酮促使人子宫肌瘤细胞周期阻滞于G₂/M期,并诱导细胞发生凋亡;左炔诺孕酮激活人子宫肌瘤细胞P53凋亡通路而引起细胞发生凋亡。结论：左炔诺孕酮引起人子宫肌瘤细胞周期阻滞于G₂/M期,并使细胞发生凋亡,进而抑制其增殖,这可能与该药物上调P53蛋白的表达,并激活Bcl-2介导的线粒体凋亡途径有关。