羽扇豆醇对人高转移肝癌HCCLM3增殖影响的机制研究

摘 要 目的： 研究羽扇豆醇（Lupeol）在人高转移肝癌细胞系HCCLM3中的抗增殖作用机制。方法: 运用CCK 8法检测不同浓度Lupeol在12-48 h对HCCLM3细胞活力的影响及相关Caspase参与Lupeol（20～100 μmol·L^-1）诱导的细胞凋亡类型;运用Realtime PCR评价Lupeol对细胞内Caspase家族及Bcl 2相关基因的mRNA表达的影响;同时运用流式细胞术检测Lupeol对细胞周期分布的影响。结果:Lupeol在24～48 h能够抑制HCCLM3的细胞增殖,并呈现浓度依赖性,在24 h处理细胞的IC₅₀为93 μmol·L^-1;运用60～100 μmol·L^-1的Lupeol能够使HCCLM3细胞在G2/M期细胞数增加1倍;Lupeol能够活化Caspase通路,与对照组相比Lupeol处理后细胞内Caspase 3的mRNA表达量增加50%～150%,同时100 μmol·L^-1的Lupeol处理细胞后使胞内p53及Bax的mRNA表达量分别上调1倍以上,并显著降低Bcl 2及PARP的mRNA水平(P<0.05或P<0.01)。结论: Lupeol具有抑制肝癌细胞增殖能力,对肝癌预防及治疗可能具有一定的协同作用。     

青蒿琥酯对人白血病K562，K562 /ADM细胞凋亡的作用及对NF-κB p65表达的影响

摘 要 目的:观察青蒿琥酯对人白血病K562、K562 /ADM细胞凋亡以及对p65表达的影响。方法: 采用流式细胞仪检测青蒿琥酯在不同浓度和不同时间段对K562、K562 /ADM细胞凋亡的影响,采用Western blot法检测15 μmol·L^-1青蒿琥酯在不同时间对K562细胞NF-κB p65表达的影响。结果 青蒿琥酯对K562细胞凋亡影响不明显,但对阿霉素耐药K562 /ADM细胞影响较大,青蒿琥浓度为7.5 μmol·L^-1和15 μmol·L^-1时,K562 /ADM细胞的调亡率明显高于K562细胞（P<0.01）,15 μmol·L^-1青蒿琥酯作用后4 h后,K562 /ADM细胞的调亡率明显增加（P<0.01）;经15 μmol·L^-1青蒿琥酯作用后, p65表达随时间的增加而明显降低（P<0.01）。结论 青蒿琥酯通过下调P65的表达而诱导白血病细胞凋亡。     

青蒿琥酯对人源肝星状细胞中Akt/GSK-3β/β-catenin信号通路的影响研究

摘 要 目的：探讨青蒿琥酯（Art）对Akt/GSK 3β/β catenin信号通路的影响。方法: 不同浓度（0,12.5,25,50 μg·mL^-1）Art作用于人源肝星状细胞（LX 2）,采用CCK 8法检测细胞增殖情况,并确定给药浓度;给予不同浓度Akt抑制药MK 2206 0～8 μmol·L^-1,Western blot法确定其最佳抑制浓度;给予Art、MK 2206、MK 2206+Art,采用Western blot法检测各组Akt、p Akt、GSK 3β、p GSK 3β、β catenin蛋白表达情况。结果: CCK 8法检测细胞存活率,当选用25 μg·mL^-1Art作用于LX 2细胞24h时细胞存活率约80%,Western blot法确定当MK 2206浓度为6 μmol·L^-1时,可有效抑制p Akt的表达;Art组（25 μg·mL^-1）、MK 2206组（6 μmol·L^-1）、MK 2206（6 μmol·L^-1）+Art（25 μg·mL^-1）组与对照组相比,Akt、p Akt、p GSK 3β、β catenin蛋白表达均有显著差异（P<0.05）,MK 2206（6 μmol·L^-1）+Art（25 μg·mL^-1）组分别与Art（25 μg·mL^-1）组、MK 2206（6 μmol·L^-1）组比较,GSK 3β、Akt蛋白表达无显著差异（P>0.05）,p Akt、p GSK 3β、β catenin蛋白表达显著降低（P<0.01）。结论: Art可通过Akt因子对Wnt/β catenin信号通路中相关因子产生影响,进而抑制细胞增殖,缓解肝纤维发展进程。     

联苯双酯对大鼠肝星状细胞增殖和凋亡及PPARγ表达的影响

摘 要 目的： 探讨联苯双酯(dimethyl dicarboxylate biphenyl,DDB)对大鼠肝星状细胞HSC-T6增殖和凋亡及过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma, PPARγ)表达的影响。方法: 接种HSC T6细胞于96孔板和6孔板中,分别用CCK 8法和流式细胞术测定不同浓度联苯双酯作用于细胞24 h后对细胞增殖和凋亡的影响;实时荧光定量PCR（Quantitative Real time PCR,Q-PCR）和Western blotting分别检测药物对HSC-T6细胞PPARγ mRNA和蛋白的表达的影响。结果: 相比于对照组（0 μmol·L^-1）,DDB在实验浓度（8～64 μmol·L^-1）能明显抑制HSC T6的增殖（P<0.05),促进HSC T6的凋亡（P<0.05);药物处理过的HSC T6细胞PPARγ mRNA及其蛋白表达均有显著提高。结论:DDB可通过上调HSC T6细胞中PPARγ的表达抑制细胞的增殖,促进细胞的凋亡。     

p53/miR-34a通路介导羽扇豆醇的抑制人膀胱癌T24细胞增殖作用

摘 要 目的：探讨羽扇豆醇对人膀胱癌T24细胞增殖的影响及对p53/miR-34a通路调控机制。方法: 运用CCK-8法检测不同浓度羽扇豆醇（5~120 μmol·L^-1）分别作用24 h和48 h对T24细胞增殖的影响;运用CCK-8法结合Caspase抑制药确证参与羽扇豆醇诱导细胞死亡的Caspase亚型;分别运用荧光定量PCR（qPCR）和蛋白免疫印记评价羽扇豆醇对miR-34a及p53蛋白表达的影响;运用qPCR评价羽扇豆醇对miR-34a下游靶基因Bcl-2、CD44、c-Myc的mRNA表达的影响。结果: T24细胞经羽扇豆醇处理后,其细胞增殖受到明显抑制,且呈一定的剂量依赖性;药物作用24 h和48 h时羽扇豆醇的半抑制浓度（IC₅₀）分别为（77.23±6.78）,（64.58±4.23）μmol·L^-1。与对照组相比,羽扇豆醇能够使T24细胞中p53蛋白表达上调,还能够增加miR-34a表达水平,差异具有统计学意义（P<0.01）;羽扇豆醇处理后细胞内Bcl-2、CD44、c-Myc的mRNA表达量下调,差异具有统计学意义（P<0.01）。结论:羽扇豆醇具有抑制膀胱癌T24细胞增殖能力,其作用机制与调控p53/miR-34a通路有关。     

吴茱萸碱对心肌细胞缺血损伤的研究

摘 要 目的：探讨吴茱萸碱对心肌细胞缺血损伤的保护作用。方法: 培养H9c2心肌细胞,缺氧24 h造成心肌细胞损伤模型,给予不同浓度（0.1,1,5,10 μmol·L^-1 ）吴茱萸预处理细胞12 h,CCK 8检测心肌细胞的活性,RT PCR检测心肌细胞炎症因子的转录,Tunel染色检测心肌细胞凋亡,免疫印迹检测信号通路的改变。结果:4种浓度的吴茱萸碱在基础状态下并未影响心肌细胞活性（P＞0.05）;缺氧24 h后,心肌细胞活性显著降低,给予1,5,10 μmol·L^-1 的吴茱萸与对照组相比差异有统计学意义（P＜0.05）,且其效果呈现剂量依耐性;细胞促炎症因子肿瘤坏死因子α（TNF α）、白细胞介素 1（IL 1）和白细胞介素 6（IL 6）的转录明显增加,心肌细胞凋亡增多,吴茱萸碱可以增加缺氧损伤的细胞活性,减少细胞炎症因子的转录,减少细胞凋亡数量,给予1 μmol·L^-1 和10 μmol·L^-1 的吴茱萸与对照组相比差异有统计学意义（P＜0.05）。免疫印迹结果显示,吴茱萸碱通过增加蛋白激酶B（AKT）和AMP依赖蛋白激酶α（AMPKα）的活性,抑制核因子 κB（NF κB）的活性发挥其心肌保护作用,给予1 μmol·L^-1 和10 μmol·L^-1 的吴茱萸与对照组相比差异有统计学意义（P＜0.05）。结论:吴茱萸碱能保护心肌细胞缺血损伤,可能成为新的抗心肌缺血药物。     

多巴胺下调 XIAP表达促进胶质瘤U251细胞凋亡的实验研究

摘 要 目的：探究多巴胺（DA）通过下调X连锁凋亡抑制蛋白（XIAP）对胶质瘤U251细胞凋亡的影响。 方法: DA处理胶质瘤U251细胞,Cell Counting Kit 8（CCK 8）法检测细胞增殖情况;线粒体膜电位（MMP）检测细胞凋亡情况;流式细胞术检测细胞凋亡率;实时荧光定量多聚酶链式反应（qRT PCR）和蛋白免疫印迹检测XIAP、B淋巴细胞瘤 2基因（BCL2）、Bcl 2相关X蛋白（BAX）、生存素（survivin）、活化的天冬氨酸特异性半胱氨酸蛋白酶（cleaved caspase3）表达情况。 结果: 与U251组相比,U251+100 μmol·L-1DA组、U251+50 μmol·L-1DA组、U251+100 μmol·L-1DA组的细胞增殖抑制率、凋亡率、BAX、cleaved caspase3 mRNA和蛋白表达量均升高（P＜0.05）,红绿荧光相对比例、XIAP、BCL2、survivin mRNA和蛋白表达量、BCL2/BAX比值均降低（P＜0.05）。与U251+25 μmol·L-1DA组相比,U251+50 μmol·L-1DA组、U251+100 μmol·L-1DA组的细胞增殖抑制率、凋亡率、BAX、cleaved caspase3 mRNA和蛋白表达量均升高（P＜0.05）,红绿荧光相对比例、XIAP、BCL2、survivin mRNA和蛋白表达量、BCL2/BAX比值均降低（P＜0.05）。与U251+50 μmol·L-1DA组相比, U251+100 μmol·L-1 DA组的细胞增殖抑制率、凋亡率、BAX、cleaved caspase3 mRNA和蛋白表达量均显著升高（P＜0.05）,红绿荧光相对比例、XIAP、BCL2、survivin mRNA和蛋白表达量、BCL2/BAX比值均显著降低（P＜0.05）。 结论: DA可能通过下调XIAP表达,从而促进胶质瘤U251细胞凋亡。     

鱼藤素降低小鼠神经母细胞瘤细胞Neuro-2A存活率的实验研究

摘 要 目的： 研究鱼藤素对小鼠神经母细胞瘤细胞Neuro-2A (N2A)存活率及凋亡的影响。方法: N2A细胞以5×10⁴·ml^-1密度接种到细胞培养板中,1×10^-8 mol·L^-1鱼藤素处理后6,12,24,48,72 h等5个时间点上测定细胞存活率;加入鱼藤素0,1×10^-11,1×10^-10,1×10^-9,1×10^-8,1×10^-7,1×10^-6 mol·L^-1等7个不同浓度干预48 h后测定细胞存活率。2×10^-8 mol·L^-1鱼藤素干预24 h,测定细胞匀浆中caspase 3活力。结果:5×10⁴·ml^-1 N2A细胞以含10%胎牛血清DMEM培养基培养,第48 h达到细胞生长曲线的峰值。1×10^-8 mol·L^-1鱼藤素干预24～72 h,时间依赖性显著降低N2A细胞存活率(P<0.05);在鱼藤素干预48 h时间点上,1×10^-8～1×10^-6·mol·L^-1浓度范围内剂量依赖性可显著降低N2A细胞存活率(P<0.05),其IC₅₀=1.6×10^-8 mol·L^-1。鱼藤素作用于N2A细胞24 h可显著提高caspase 3活力,约达到对照组的5.6倍。结论: 鱼藤素可时间依赖性且剂量依赖性降低N2A细胞存活率,可能与增加caspase 3活力有关。     

离子色谱法测定枸橼酸氢钾钠颗粒中多种成分的含量

摘 要 目的：建立离子色谱法测定枸橼酸氢钾钠颗粒中钠、钾和枸橼酸含量的方法。方法: 钾和钠的色谱条件：采用Dionex IonPac CS12A色谱柱（250 mm×4.6 mm,5 μm）,流动相为0.02 mol·L^-1甲烷磺酸溶液,流速为1.0 ml·min^-1,抑制器为CSRS 300,抑制电流为59 mA,采用抑制型电导检测器,进样量为25 μl。枸橼酸的色谱条件：采用Dionex HPICE AS1离子排斥色谱柱（250 mm×9.0 mm,7.5 μm）,流动相为0.015 mol·L^-1硫酸溶液,流速为0.6 ml·min^-1,检测波长为220 nm,进样量为10 μl。结果: 钠的线性范围为0.82～82.49 μg·ml^-1（r=0.999 9）,平均回收率为98.9%,RSD为0.55% （n=9）;钾的线性范围为1.38～137.89 μg·ml^-1（r=1.000 0）,平均回收率为100.5%,RSD为0.53%（n=9）;枸橼酸的线性范围为0.021～10.600 mg·ml^-1（r=1.000 0）,平均回收率为99.1%,RSD为0.54%（n=9）。结论:本方法简便、快速、准确,可用于枸橼酸氢钾钠颗粒的质量控制。     

HPLC法测定追风舒筋活血片中马钱子碱、士的宁的含量

摘 要 目的： 建立追风舒筋活血片中马钱子碱、士的宁的含量测定方法。方法: 应用高效液相色谱法测定,色谱柱为Agilent SB C₁₈柱(250 mm×4.6 mm,5 μm),以乙腈 0.01 mol·L^-1庚烷磺酸钠与0.02 mol·L^-1磷酸二氢钾等量混合溶液（用10%磷酸调节pH至2.8）（21∶79）为流动相,检测波长为260 nm,流速为1.0 ml·min^-1,柱温为35℃,进样量为10 μl。结果: 马钱子碱和士的宁的线性范围分别为0. 011 0~0.219 6 mg·ml^-1 (r=0.999 4)、0.010 1~0.202 8 mg·ml^-1 (r=0.999 7);平均加样回收率分别为98.24%(RSD=1.54%)、97.92%(RSD=1.49%)(n=6) 。结论:该方法简便易行、准确、重复性好,可用于追风舒筋活血片的质量控制。     

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.