槲皮素对肝癌HepG2细胞生长影响的体外研究

目的 观察槲皮素对肝癌HepG2细胞生长及hTERT基因表达的影响.方法 以台盼蓝拒染法计数肝癌细胞的生长抑制率,用透射电镜从形态变化方面了解凋亡的发生,流式细胞术检测细胞周期变化,Western-blot检测hTERT基因表达改变,PCR-TRAP法检测端粒酶活性.结果 槲皮素抑制肝癌HepG2细胞增殖的作用明显,且呈浓度和时间依赖性,槲皮素处理48 h后的半数抑药浓度（IC5o）为25.5μmol/L;形态学检测显示出了细胞凋亡的特征变化,经10-20 μm/L的槲皮素处理,肝癌HepG2细胞周期阻滞于G0/G1期,且HepG2细胞hTERT蛋白降低,端粒酶活性被抑制.结论 槲皮素能呈时间、剂量依赖性地抑制肝癌细胞的生长,能诱导HepG2细胞发生凋亡,其抑制增生与诱导凋亡的机制可能与下调hTERT基因表达、抑制端粒酶活性、破坏端粒稳定性有关.     

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。     

紫杉醇体外诱导人胃癌细胞凋亡及对端粒酶活性的影响

目的　研究紫杉醇对人胃癌细胞诱导凋亡作用及端粒酶活性变化。方法　 0 .0 0 1～ 1μmol/L的紫杉醇处理SGC 790 1细胞后 ,用MTT法测定胃癌细胞的生长抑制率 ,通过形态学观察及流式细胞术检测细胞凋亡率 ,半定量TRAP -银染法测定端粒酶活性变化。结果　不同浓度的紫杉醇对胃癌细胞均有明显抑制作用 ,且呈时间依赖性及剂量依赖性 ,光镜及流式细胞术分析表明 ,0 .0 1μmol/L的紫杉醇处理后 2 4h ,细胞即出现明显的凋亡形态特征及凋亡峰 ,对端粒酶活性的同步检测结果显示 ,紫杉醇在诱导细胞凋亡的同时伴随端粒酶活性下调 ,且随紫杉醇浓度增大 ,抑制作用逐渐增强 ,2 4h酶活性即显著受抑制 ,72h变为阴性。结论　紫杉醇对胃癌细胞具有明显抑制作用 ,诱导细胞凋亡并抑制端粒酶活性可能是其发挥抗癌作用的机制之一 ,端粒酶可作为肿瘤化疗的敏感性指标。     

As_2O_3诱导MGC803细胞凋亡中TRF1、TRF2蛋白表达及端粒稳定性的作用

目的分析三氧化二砷(As2O3)对人胃癌MGC803细胞端粒重复序列结合因子1、2(TRF1、TRF2)表达及端粒稳定性的影响,探讨As2O3诱导细胞凋亡的机制。方法体外培养MGC803细胞,MTT比色实验分析As2O3对MGC803细胞增殖抑制作用,流式细胞术检测MGC803细胞凋亡,进行染色体端-端融合分析及Westernblot检测TRF1、TRF2蛋白表达。结果MTT法显示As2O3对人胃癌MGC803细胞有明显的抑制作用,且呈时间、剂量依赖关系。流式细胞术检测出现典型的凋亡峰,并随As2O3浓度增加和作用时间延长而增高,对照组未见此改变。染色体端-端融合分析显示5μmol·L-1As2O3处理MGC803细胞48h染色体融和率明显高于对照组。Westernblot分析结果表明TRF1在5μmol·L-1As2O3处理MGC803细胞48h后,表达上升,而TRF2表达下降。结论实验结果提示As2O3通过下调TRF2蛋白表达、上调TRF1蛋白表达及使染色体端-端融合,从而诱导MGC803细胞凋亡。     

三氧化二砷对人肝癌细胞凋亡及Bcl-2、caspase-3蛋白表达的影响

目的研究三氧化二砷（arsenic trioxide,As2O3）诱导HepG2细胞凋亡及其机制。方法采用MTT法、形态学观察及流式细胞术方法检测As2O3对HepG2细胞的生长抑制、凋亡作用的影响,用Western blot检测As2O3处理后HepG2细胞内凋亡相关蛋白Bcl-2和caspase-3的表达。结果As2O3对HepG2细胞有明显的抑制增殖作用,且呈浓度和时间依赖性,细胞呈典型的凋亡形态学改变,As2O3呈时间依赖性诱导HepG2细胞凋亡。As2O3处理不同时间后,Bcl-2蛋白表达水平下调,caspase-3蛋白表达水平升高。结论As2O3具有诱导人肝癌HepG2细胞凋亡的作用,其机制可能与其下调Bcl-2蛋白的表达,促进caspase-3活化有关。     

hTERT反义寡核苷酸增强DDP介导细胞凋亡作用

目的研究端粒酶催化亚基(hTERT)反义寡核苷酸(ASODN)对HeLa细胞端粒酶活性的抑制及其对顺铂(DDP)诱导细胞凋亡的影响。方法用逆转录聚合酶链反应技术(RT-PCR)定量端粒酶重复扩增法(TRAP)检测细胞的端粒酶活性。观察细胞凋亡的形态学变化,流式细胞仪对细胞凋亡进行定量分析。结果ASODN作用后,细胞的端粒酶活性明显降低,且这种作用具有明显的时间和剂量依赖性。0.05、0.1μmol.L-1和0.2μmol.L-1的硫代ASODN治疗后,HeLa细胞的端粒酶活性分别下降了21.8%、52.4%和71.1%。0.2μmol.L-1的硫代ASODN作用HeLa24、48h和72h后,细胞的端粒酶活性分别下降了12.48%、38.27%和71.10%。细胞转染0.2μmol.L-1浓度的ASODN24h后再与1.5、3.0mg.L-1浓度的顺铂联合作用,吖啶橙染色可见典型的凋亡形态,并且凋亡百分率(50.35%、29.67%)分别与RSODN联合顺铂组(19.33%、12.13%)、单用顺铂组(19.67%、11.38%)比较,差异有显著性(P<0.05)。结论hTERT反义寡核苷酸能有效抑制其端粒酶活性,并且促进DDP诱导HeLa细胞凋亡。     

三氧化二砷对恶性淋巴瘤细胞株的作用及临床研究初探

目的：研究不同浓度的三氧化二砷（As2O3)对恶性淋巴瘤细胞系和淋巴瘤患者活体淋巴瘤细胞作用及其初步临床应用效果。方法：用MTT法检测不同浓度的As2O3对Daudi,Raji及活体细胞株生长的影响，用电镜及流式细胞仪检测As2O3诱导淋巴瘤细胞凋亡情况。12例Ⅲ-Ⅳ期的非霍奇金淋巴瘤患者，给予0．1^ As2O310 mL/d,30d为1个疗程，缓解后巩固治疗采用原方案每月连用7d，结果：1．0－2．0μmol/L的As2O3可明显降低Daudi,Raji及活体细胞株活力，可见明显凋亡小体；用流式细胞仪检测出现DNA含量低于G1期的亚G1期细胞，在G1／G0期前出现凋亡峰，其数量与药物作用时间呈正相关。12例患者单用As2O3治疗1个疗程，完全缓解1例，部分缓解2例，稳定6例，均无骨髓抑制及其它副作用等。结论：一定浓度的As2O3显著抑制3株淋巴瘤细胞的生长，诱导凋亡。AS2O3对一般化疗无效者，仍有一定疗效。     

三氧化二砷诱导喉癌细胞凋亡的体外研究

目的探讨三氧化二砷(As2O3)诱导喉癌Hep-2细胞株凋亡的作用。方法体外培养的Hep-2细胞与不同浓度的As2O3作用不同时间,用流式细胞仪、DNA琼脂糖凝胶电泳研究细胞凋亡的发生及对细胞周期的影响。结果流式细胞仪检测发现不同药物浓度和作用时间下均有细胞凋亡发生。2滋mol/LAs2O3引起细胞周期明显变化,作用24h后S期细胞比例增高,72h后S期细胞明显下降,细胞大量凋亡。结论As2O3可有效抑制人喉癌Hep-2细胞的体外生长,具有时间、浓度依赖性。As2O3诱导Hep-2细胞凋亡主要是S期的细胞。     

三氧化二砷对人肺癌A549细胞凋亡及耐药基因表达的影响

目的研究三氧化二砷（As2O3）对人肺癌细胞株的诱导凋亡作用及对耐药基因LRP表达的影响。方法应用人肺腺癌A549细胞株,运用体外细胞培养法、MTT法、流式细胞术检测As2O3对人肺癌细胞株抑制及诱导凋亡作用;用逆转录-聚合酶链反应（RT-PCR）方法检测LRP mRNA的表达。结果As2O3对人肺癌A549细胞具有抑制作用,其抑制率呈时间-剂量依赖关系。不同浓度的As2O3均可诱导凋亡。1．0μmol／L、3．0μmol／L的As2O3可下调LRPmRNA的表达。结论As2O3具有抑制肿瘤细胞生长及诱导细胞凋亡作用,其机制与下调LRPmRNA表达有密切关系。     

四种砷拮抗剂对三氧化二坤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

目的：研究巯基供体N-乙酰兰胱氨酸（NAC）和二巯丁二钠（NDMS）、抗氧化剂过氧化氢酶（CAT）和Ca^2 清除剂（Quin2）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Quin2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。NAC4mmol/L,NDMS200μmol/L,CAT80kU/L,Quin 2 20μmol/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Quin2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Quin2可不同程度拮抗三氧化二砷对三种细胞的作用。     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Aquatic Insects and Trace Metals: Bioavailability,Bioaccumulation, and Toxicity

Abstract

The uptake of metals from food and water sources by insects is thought to be additive. For a given metal, the proportions taken up from water and food will depend both on the bioavailable concentration of the metal associated with each source and the mechanism and rate by which the metal enters the insect. Attempts to correlate insect trace metal concentrations with the trophic level of insects should be made with a knowledge of the feeding relationships of the individual taxa concerned. Pathways for the uptake of essential metals, such as copper and zinc, exist at the cellular level, and other nonessential metals, such as cadmium, also appear to enter via these routes. Within cells, trace metals can be bound to proteins or stored in granules. The internal distribution of metals among body tissues is very heterogeneous, and distribution patterns tend to be both metal and taxon specific. Trace metals associated with insects can be both bound on the surface of their chitinous exoskeleton and incorporated into body tissues. The quantities of trace meals accumulated by an individual reflect the net balance between the rate of metal influx from both dissolved and particulate sources and the rate of metal efflux from the organism. The toxicity of metals has been demonstrated at all levels of biological organization: cell, tissue, individual, population, and community. Much of the literature pertaining to the toxic effects of metals on aquatic insects is based on laboratory observations and, as such, it is difficult to extrapolate the data to insects in nature. The few experimental studies in nature suggest that trace metal contaminants can affect both the distribution and the abundance of aquatic insects. Insects have a largely unexploited potential as biomonitors of metal contamination in nature. A better understanding of the physico-chemical and biological mechanisms mediating trace metal bioavailability and exchange will facilitate the development of general predictive models relating trace metal concentrations in insects to those in their environment. Such models will facilitate the use of insects as contaminant biomonitors.     

Alzheimer’s disease – current trends in basic and clinical research. Highlights from the 16th ECNP

Advances in the molecular biological knowledge of neuronal nicotinic acetylcholine receptors (nAChRs) have led to a growing interest by the pharmaceutical industry in the development of novel compounds that selectively modulate nAChR function. The ability of (-)-nicotine, an activator of nAChRs, to enhance attentional aspects of cognition in animals and humans, to exert neuroprotective and anxiolytic-like effects, and presumably to mediate the negative correlation between smoking and Alzheimer's (and Parkinson's) Disease, has focused interest on the potential therapeutic utility of modulators of nAChR function for treatment of some of the deficits associated with these progressive, neurodegenerative conditions. Numerous compounds are known which activate nAChRs and which might serve as lead compounds toward the development of such agents. The pharmacologic diversity of neuronal nAChR subtypes suggests the possibility of developing selective compounds which would have more favourable side-effect profiles than existing agents. This broader class of agents, collectively called cholinergic channel modulators (ChCMs), is anticipated to encompass compounds which would have more favourable side-effect profiles than existing agents, which generally exhibit low selectivity. This selectivity may be achieved by preferentially activating some subtypes of nAChRs (i.e., Cholinergic Channel Activators, ChCAs) or inhibiting the function of other subtypes (Cholinergic Channel Inhibitors, ChCIs). An overview of the biology of nAChRs and the rationale for the use of ChCMs for the treatment of dementia related to neurodegenerative diseases are presented, followed by a discussion of lead compounds and compounds under consideration for clinical evaluation.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.