SMYD3调控人乳腺癌MCF-7细胞增殖机制的研究

目的：通过转染技术抑制或增强SMYD3基因的表达,以探讨SMYD3对乳腺癌MCF-7细胞增殖的调控机制机制。方法：以MTY法和软琼脂克隆形成实验研究抑制或增强SMYD3基因表达对细胞增殖的影响,以RT-PCR法和Western印迹法检测MCF-7细胞经转染后,胞内SMYD3、ERK／MAPK通路相关蛋白及其磷酸化产物,以及凋亡和细胞周期调控相关蛋白表达水平的变化。结果：用针对SMYD3基因的shRNAs表达质粒转染MCF-7细胞后,其SMYD3基因mRNA和蛋白表达水平下调,细胞生长受到抑制,细胞中ERK1／2、CyclinD1和CDK4蛋白水平明显下调;软琼脂克隆形成实验显示,增强SMYD3基因表达能明显促进MCF-7细胞克隆形成。结论：SMYD3可通过激活ERK／MAPK信号转导通路和上调细胞周期相关蛋白CyclinD1和CDK4的水平提高肿瘤细胞的增殖能力。     

丹参酮ⅡA逆转人ER阳性乳腺癌细胞的恶性表型及其机理

目的 证实丹参酮ⅡA对体外培养的雌激素受体(ER)阳性人乳腺癌细胞(MCF-7)的增殖抑制及恶性表型的逆转作用;探讨其作用的分子机理.方法 用光镜、电镜观察丹参酮ⅡA处理前后细胞形态的改变;细胞增殖试验(MTT法)、集落形成试验检测丹参酮ⅡA对MCF-7细胞生长、增殖的影响;RT-PCR技术半定量检测细胞增殖、分化相关基因mRNA表达的改变;流式细胞仪定量检测细胞增殖、分化相关基因蛋白表达的改变.结果 经一定浓度丹参酮ⅡA处理后MCF-7细胞形态改变,接近正常上皮细胞形态;丹参酮ⅡA对MCF-7细胞增殖抑制作用具有明显的剂量一时问依赖关系;对MCF-7细胞增殖具抑制作用;可上调增殖、分化相关基因ERa、nm23-1、c-fos的表达,下调c-myc的表达.结论 丹参酮ⅡA对MCF-7细胞体外生长、增殖有明显抑制作用;对MCF-7细胞形态具有恶性表型逆转作用;可上调细胞ERa、c-fos、nm23-1和下调c-myc基因表达,可能通过调控细胞增殖和分化相关基因的表达来逆转肿瘤细胞的恶性表型.     

姜黄素诱导乳腺癌MCF-7细胞凋亡

目的 研究姜黄素对人乳腺癌细胞株MCF-7细胞增殖抑制和诱导凋亡作用.方法 MTT法检测姜黄素对MCF-7细胞的增殖抑制作用;流式细胞术(FCM)PI单染检测细胞周期;Annexin V/PI双染法检测细胞凋亡;Western blot法检测Bcl-2和Bax蛋白的表达.结果 姜黄素对MCF-7细胞生长有明显抑制作用,并呈剂量、时间依赖性;姜黄素能使MCF-7细胞阻滞在G1/S期,可以诱导细胞凋亡,Bax蛋白表达上调,而Bcl-2的表达减少.结论 姜黄素对人乳腺癌MCF-7细胞的增殖具有显著的抑制作用并可诱导细胞凋亡.其分子作用机制可能与其上调Bax基因表达水平的同时下调Bcl-2基因表达水平,从而诱导细胞凋亡有关.     

三氧化二砷诱导NB4细胞的基因表达谱改变

目的：研究三氧化二砷在诱导急性早幼粒细胞白血病细胞株NB4分化过程中基因表达谱的改变．方法：通过逆转录方法将经过及未经过三氧化二砷处理的NB4细胞mRNA制备成两种探针,应用Cy3和Cy5两种荧光染料分别标记这两种探针,随后与包含1003条待研究人类基因的表达谱基因芯片杂交,通过扫描和计算机软件分析,寻找经三氧化二砷作用后表达有差异的基因．结果：NB4细胞在三氧化二砷(0．5μmol／L)作用后3条基因上调,18条基因下调．1条参与蛋白酶体降解途径的基因显著上调,多条与细胞信号传导、RNA加工及蛋白质合成相关基因下调．结论：PSMB6及ITGBI基因的表达改变可能与NB4细胞的凋亡和／或分化有密切关系．     

姜黄素对人乳腺癌MCF-7细胞增殖及血管生成相关基因表达的影响

目的研究姜黄素对人乳腺癌MCF-7细胞增殖的抑制作用,并研究对血管生成相关基因mRNA表达的影响,探讨姜黄素抑癌可能的分子机理。方法体外培养人MCF-7细胞,细胞分为姜黄素高、中、低剂量组、空白对照组、溶剂对照组。采用MTT法检测姜黄素对人MCF7细胞增殖的抑制作用;应用RT-PCR检测各组细胞血管生成相关基因ANGPTL3、VEGF mRNA的表达。结果①姜黄素对MCF-7细胞的生长有明显的抑制作用。②经姜黄素处理后MCF-7细胞的ANGPTL3、VEGF的mRNA表达水平下调。结论①姜黄素在体外对MCF7细胞生长具有抑制作用。②姜黄素能显著下调MCF-7细胞血管生成相关基因ANGPTL3、VEGF mRNA的表达。③姜黄素对人MCF-7细胞增殖的抑制效应可能与ANGPTL3、VEGF下调有关。     

乙酰氧基胡椒酚乙酸酯对人乳腺癌MCF-7细胞凋亡与侵袭的影响

目的:研究乙酰氧基胡椒酚乙酸酯(ACA)对人乳腺癌MCF-7细胞凋亡与侵袭的影响。方法:通过RT-PCR法检测ACA对人乳腺癌MCF-7细胞B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)基因表达的影响,免疫细胞化学法检测细胞中基质金属蛋白酶(MMP)-2和MMP-9的表达。结果:MCF-7细胞经50、100、150μmol·L-1ACA干预后,Bcl-2 mRNA表达显著下降,Bax mRNA表达显著升高(P<0.01);50、100、150μmol·L-1ACA作用后细胞中MMP-2和MMP-9的蛋白表达显著减少(P<0.01)。结论:ACA诱导的Bcl-2 mRNA表达的下调和Bax mRNA表达的上调可能参与了其诱导人乳腺癌MCF-7细胞凋亡作用。ACA能抑制人乳腺癌MCF-7细胞中MMP-2和MMP-9的蛋白表达从而抑制其侵袭。     

FANCF基因沉默对人乳腺癌细胞生物学特性的影响

目的研究FANCF基因沉默对乳腺癌MCF-7细胞生物学特性的影响,并检测相关指标的变化,探讨FANCF基因是否参与乳腺癌的发生、发展及耐药性的形成。方法构建FANCF-shRNA质粒,转染FANCF-shRNA使乳腺癌MCF-7细胞FANCF基因沉默;MTT法检测FANCF基因沉默对MCF-7细胞增殖的影响;流式细胞仪PI单染法及AnnexinV-FITC/PI双染法检测FANCF基因沉默对MCF-7细胞周期及凋亡的影响;RT-PCR检测FANCF基因沉默前后耐药相关基因mRNA表达水平的变化。结果成功构建FANCF-shRNA质粒,RT-PCR验证转染FANCF-shRNA后48h出现FANCF基因沉默;与阴性对照组相比,FANCF基因沉默后,MCF-7细胞增殖明显受到抑制,转染FANCF-shRNA后48h和72h的抑制率分别为12.65%±1.24%和29.64%±0.87%(P<0.05);S期MCF-7细胞比率增加(33.38%±0.68%,P<0.05),细胞周期阻滞在S期;转染FANCF-shRNA后MCF-7细胞凋亡率明显增加,转染后48h和72h的凋亡率分别为28.95%±1.81%和49.00%±2.32%(P<0.05);P-gp、BCRPmRNA表达水平降低,MRP、LRPmRNA表达水平无明显变化(P>0.05)。结论FANCF基因沉默可抑制人乳腺癌MCF-7细胞增殖,促进细胞凋亡,并使细胞周期阻滞于S期;并可抑制BCRP等耐药相关基因的mRNA表达,参与乳腺癌细胞耐药性的调控。     

小檗碱和环孢素联用对大鼠肝脏肿瘤及免疫相关基因表达影响的基因芯片研究

目的:研究小檗碱和环孢素联用时大鼠肝组织中肿瘤及免疫相关基因表达谱的影响。方法:大鼠予以盐酸小檗碱ig 6 d,最后一天予以环孢素单次ig给药。取肝脏提取总RNA。用Cy3和Cy5两种荧光物质分别标记阴性时照组,环孢素组和环孢素 盐酸小檗碱组肝脏mRNA,制备成探针与表达谱芯片进行杂交。结果:在被检测的4 096个基因中,环孢素组与肿瘤及免疫相关的差异表达基因共3个,均为表达下调。环孢素 盐酸小檗碱组与肿瘤及免疫相关的盖异表达基因共14个,其中表达上调的相关基因5个,表达下调的相关基因9个。结论:小檗碱和环孢素联用可影响大鼠肝组织中肿瘤及免疫相关基因的表达。     

低剂量双酚A及其与苯并芘联合染毒对人乳腺上皮细胞MDM2基因的影响

目的观察BPA与B[a]P联合作用对3种乳腺上皮细胞MDM2及TP53基因表达的影响。方法采用细胞增殖荧光法检测环境相关剂量BPA对3种乳腺上皮细胞S期细胞比例的影响,采用实时荧光定量PCR和蛋白免疫印迹的方法分别检测环境相关剂量水平(10-9、10-7mol/L)BPA及其与10-6mol/L B[a]P联合作用对3种乳腺上皮细胞中MDM2和TP53基因及其编码的蛋白表达水平的影响。结果环境相关剂量的BPA可以引起MCF-7细胞S期细胞比例增高;环境相关剂量的BPA单独作用对3种类型乳腺上皮细胞MDM2和TP53基因表达水平无显著影响;B[a]P单独作用可以引起MCF-10A细胞中MDM2基因表达水平的轻微增加及MCF-7细胞中MDM2基因表达的显著增加;在MCF-7细胞中BPA与B[a]P联合作用可明显增加B[a]P所引起的MDM2基因表达上调,而在HMEC和MCF-10A细胞未发现该现象,蛋白水平与mRNA水平的改变基本一致,同时p53/mdm2比值降低。结论环境相关剂量的BPA与B[a]P联合作用可以在雌激素受体表达阳性的MCF-7细胞中引起MDM2基因及其蛋白产物表达水平的显著上调,提示环境暴露剂量的BPA可能通过雌激素受体依赖的作用途径增加化学致癌物致乳腺癌发生风险。     

高密度Oligo基因芯片检测榄香烯对胶质瘤U251细胞凋亡相关基因的影响

目的应用高密度Oligo基因芯片技术检测榄香烯对胶质瘤U251细胞凋亡相关基因的影响。方法实验分2组,实验组用含有终浓度为0.062mg/ml榄香烯作用于U251细胞24h,对照组为未行任何处理的U251细胞。抽提2组U251细胞的总RNA并逆转录合成Cy5、Cy3标记的cDNA,混合后杂交含22000个人类基因的人类高密度Oligo基因芯片,经过洗片和扫描,获得荧光信号图像并用计算机分析,随机抽取4种差异表达基因用PCR验证。结果按差异显著性标准,从22000条基因中筛选出差异表达基因179条,其中11个凋亡相关基因表达上调,15个凋亡相关基因表达下调。主要包括细胞周期蛋白、细胞凋亡、细胞骨架和运动蛋白等相关基因。结论榄香烯能够影响胶质瘤细胞相关凋亡基因的改变,而基因芯片技术有效分析其基因表达谱,有助于认识胶质瘤药物治疗的机制。     

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.     

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.     

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.