土贝母皂苷诱导人鼻咽癌细胞株CNE-2Z细胞凋亡的研究

目的　研究土贝母皂苷 (下称皂苷 )对人鼻咽癌细胞株CNE 2Z细胞凋亡的影响。方法　MTT法检测皂苷对CNE 2Z细胞生长的影响 ;形态学方法 (荧光显微镜和透射电镜 )、流式细胞仪和DNA琼脂糖凝胶电泳观察和分析在皂苷作用下CNE 2Z细胞形态、DNA含量的变化和DNA断裂的情况 ;蛋白免疫印迹法检测凋亡相关基因bcl 2、bax和cas pase 3表达的变化。 结果　皂苷抑制CNE 2Z细胞的生长 ,其效果与皂苷的浓度和作用时间相关 ;诱导CNE 2Z细胞发生典型程序性死亡 ;凋亡抑制基因bcl 2表达逐渐减少 ;而凋亡诱导基因bax和caspase 3在 1、3、5h表达增高。 结论　诱导细胞凋亡是皂苷发挥抗瘤效果的一种重要机制 ;皂苷诱导的CNE 2Z细胞凋亡与bcl 2、bax和caspase 3基因有关     

去甲斑蝥素对体外微管蛋白聚合的抑制作用

目的探讨去甲斑蝥素(NCTD)对微管蛋白聚合-解聚过程的影响。方法采用蛋白凝胶电泳鉴定从猪脑中提取的微管蛋白,并用Bradford法测定微管蛋白含量。在经过秋水仙碱和紫杉醇验证稳定的37℃和4℃平衡体系内加入NCTD 10,20和30μmol.L-1观察微管蛋白聚合-解聚活性。人卵巢癌SK-OV-3细胞加入NCTD 60μmol.L-1作用8 h,取上清和沉淀,Western印迹法检测微管蛋白含量。结果与空白对照组相比,在37℃聚合反应中,NCTD 10,20和30μmol.L-1组对微管蛋白聚合的抑制率分别为(5.5±2.7)%,(7.1±1.2)%和(27.5±0.4)%,在此浓度范围内NCTD随浓度的升高抑制作用增强(P<0.05),但对微管蛋白在4℃的解聚影响不显著。NCTD 60μmol.L-1作用人卵巢癌细胞后,细胞中游离微管蛋白增加了(91.5±8.5)%,聚合微管蛋白量减少了(51.8±3.8)%,说明NCTD抑制了人卵巢癌细胞SK-OV-3中微管蛋白的聚合。结论 NCTD对体外微管蛋白的聚合具有一定的抑制作用。     

新藤黄酸通过内质网应激诱导人鼻咽癌 CNE-2Z细胞凋亡的研究

目的 探讨新藤黄酸(gambogenic acid,GNA)对人鼻咽癌CNE-2Z细胞的影响和相关分子机制.方法 采用倒置显微镜观察药物处理组和对照组的CNE-2Z细胞形态的变化;MTT法检测药物处理组的CNE-2Z细胞增殖的抑制作用;DAPI染色和AV/PI 双染实验观察细胞凋亡情况;蛋白质印迹法检测GRP78、CHOP和ATF4蛋白的表达.结果 倒置显微镜和荧光显微镜观察,发现与对照组相比,GNA作用CNE-2Z细胞后,体积缩小变圆,细胞核发生典型核染色质固缩等细胞凋亡形态学的变化.GNA可以抑制人鼻咽CNE-2Z细胞的增殖,并有时间和浓度依赖性.AV/PI染色后早凋细胞膜呈苹果绿色,晚凋或坏死细胞质呈不同程度的黄-红色.蛋白质印迹法检测表明GRP78蛋白表达总体呈现浓度依赖性下调,上调了CHOP蛋白和ATF4蛋白.结论 GNA可以影响人鼻咽癌CNE-2Z细胞的增殖,引发细胞凋亡,这可能与内质网应激相关蛋白(GRP78、CHOP和ATF4蛋白)有关.     

文蛤多肽抑制肿瘤细胞微管蛋白聚合

目的探讨文蛤多肽(Mere15)抑制人肺癌A549细胞增殖作用及其机制。方法 MTT比色法检测细胞增殖抑制率;流式细胞仪分析细胞周期分布的变化;微管蛋白免疫荧光检测技术检测Mere15对微管蛋白聚合的影响;Western blot检测Mere15对p21蛋白表达的影响。结果 Mere15可抑制A549细胞生长,抑制率呈剂量和时间依赖性;随着处理时间的增加,A549细胞G2/M期比例逐渐升高,微管蛋白聚合受到抑制,p21蛋白表达水平逐渐升高。结论 Mere15具有抑制A549细胞增殖的作用,其机制可能与抑制微管蛋白聚合有关。     

大蒜提取物Z-ajoene对U937细胞增殖抑制作用机制的研究

目的　研究大蒜含硫化合物Z ajoene抗肿瘤作用的分子机制。方法　应用MTT法测定Z ajoene对不同白血病细胞株的生长抑制作用。采用流式细胞技术 (FACS)分析Z ajoene对U937细胞周期的影响以及对该细胞的诱导凋亡作用。用Western blot的方法检测Z ajoene对凋亡相关蛋白以及微管蛋白表达的影响。结果　Z ajoene对两种白血病细胞系HL6 0和U937有着相近的生长抑制作用 ,IC50 值均约为10 μmol·L-1。细胞周期分析发现 ,在给药初期和药物浓度较低时G2 /M期细胞数量明显升高 ;2 0 μmol·L-1Z ajoene作用 2 4h时 ,G2 /M期细胞比对照组升高约15 2 5 %。Z ajoene可以诱导U937细胞凋亡 ,并呈浓度和时间依赖性。实验结果表明 ,细胞凋亡过程中的关键蛋白酶caspase 3的激活及其底物PARP蛋白的裂解随着给药浓度和时间的增加而增加。此外 ,Z ajoene也产生以出现 2 3ku裂解带为特征的Bcl 2裂解。另外 ,实验浓度的Z ajoene对β 微管蛋白的表达有抑制作用 ,而对α 微管蛋白表达无明显改变。结论　Z ajoene能明显抑制U937细胞株的生长并可阻断U937细胞周期于G2 /M期 ,且这种阻断早于细胞凋亡。Z ajoene抗肿瘤机制与诱导细胞凋亡和干扰微管蛋白聚合相关。     

紫杉醇抗人舌癌细胞株的体外研究

目的：观察紫杉醇对人舌癌细胞的体外作用，探讨其抗癌效果及作用机制。方法：MTT法检测紫杉醇体外抗Tca8113人舌癌细胞作用，光镜和电镜下观察受处理细胞形态学变化，并用流式细胞仪分析其细胞周期的改变。结果：MTT法和形态学观察显示紫杉醇对Tea8113人舌癌细胞增殖有明显抑制作用，流式细胞仪分析经处理的癌细胞有G2／M期阻滞和细胞凋亡现象。电镜下可见紫杉醇处理的癌细胞的微管聚合。结论：紫杉醇是一种抗微管类的抗癌药物和G2／M阻滞剂。     

以微管蛋白为靶的高通量药物筛选方法的建立与应用

目的：以微管蛋白为靶建立高通量筛选（HTS）模型，以便有效地发现抗肿瘤化合物。方法：细胞培养与免疫荧光技术。结果：以常规的免疫组化（玻片）方法为基础优化实验条件，在96孔板上建立了以微管蛋白为靶点的高通量药物筛选模型；抗肿瘤药物紫杉醇和秋水仙碱作用于人肝癌HepG2细胞后，细胞的免疫荧光强度发生了明显的可检测的变化，间接反映药物对细胞徽管蛋白聚合／解聚作用的影响，与理论预测结果一致。结论：基于人肿瘤细胞的以徽管蛋白为靶点的高通量筛选方法可用于抗肿瘤化合物的筛选。     

普鲁卡因对鼻咽癌细胞p16蛋白表达的影响

目的探讨普鲁卡因(procaine,PCA)对人鼻咽癌细胞CNE-2Zp16蛋白表达的影响。方法应用MTT法检测不同浓度PCA对人鼻咽癌细胞CNE-2Z增殖的影响,应用免疫组化法测定鼻咽癌细胞p16蛋白的表达。结果PCA能显著抑制CNE-2Z细胞增殖,该作用呈剂量-效应和时间依赖关系。PCA组CNE-2Z细胞p16蛋白表达明显高于对照组。结论PCA对CNE-2Z细胞增殖有抑制作用,它能上调CNE-2Z细胞p16蛋白的表达,推测这可能是PCA抑制CNE-2Z细胞增殖的重要分子机制之一。     

机械牵张对缺血缺氧心肌细胞微管的影响

目的：探讨机械牵张对缺血缺氧心肌细胞微管的影响。方法：建立离体培养的SD乳鼠心肌细胞机械牵张模型，施加无糖缺氧刺激因素模拟烧伤早期缺血缺氧损害．实验分为10％牵张组（S组）、缺血缺氧组（Ⅰ组）及10％牵张＋缺血缺氧组（IS组），分别在刺激前和后1、3、6、12小时4个时相点进行观察，采用免疫荧光染色观察微管的变化，并用免疫蛋白印迹检测β-tubulin含量变化。结果：3小时后S组微管呈现增强趋势，Ⅰ组微管有明显破坏，可见大量断裂微管分布于核周，IS组微管破坏严重，无聚合状态微管存在，细胞完整性受损。刺激后S组β-tubulin含量逐渐增多，Ⅰ组则减少．IS组减少显著。12小时后IS组与其他2组差异显著（P〈0．01）。结论：机械牵张加重了缺血缺氧对心肌细胞微管的破坏，可能是“休克心”发生发展的重要原因之一。     

丹酚酸C诱导肝癌HepG2细胞有丝分裂阻滞及凋亡的研究

目的研究丹酚酸C(salvianolic acid C,SalC)的体外抗肿瘤细胞增殖作用及其分子机制。方法 MTT法检测Sal C对人肿瘤细胞的增殖抑制作用,流式细胞术检测细胞周期分布及细胞凋亡,Giemsa染色进行细胞有丝分裂的形态学观察,微管蛋白聚合实验检测Sal C对微管蛋白聚合活性的影响,比色法检测细胞中Caspase-3和Caspase-6的活性。结果丹酚酸C能抑制多种人肿瘤细胞增殖,其中对HepG2细胞的抑制作用较明显。流式细胞术分析发现Sal C使HepG2细胞阻滞于G2/M期并随作用时间延长出现明显的凋亡峰,细胞中Caspase-3和Caspase-6的活性升高。Gi-emsa染色提示HepG2细胞发生有丝分裂阻滞。Sal C能明显抑制微管蛋白的聚合。结论 Sal C具有抗肿瘤细胞增殖活性,通过抑制微管蛋白聚合诱导细胞有丝分裂阻滞从而诱导凋亡。     

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.     

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.     

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.