马齿苋总黄酮对血小板源性生长因子致家兔血管平滑肌细胞增殖的抑制作用

目的：研究马齿苋总黄酮（Portulaca totalflavone,PTF）对血小板源性生长因子-BB型（PDGF-BB）致血管平滑肌细胞（Vascularsmoothmuscle cell,VSMC）增殖的影响并探讨其机制。方法：采用组织贴壁法培养家兔胸主动脉血管平滑肌细胞,细胞计数法观察PTF（8、24、72mg/L）对PDGF-BB所致的VSMC增殖作用的影响;氚-胸腺嘧啶核苷（3H-TdR）掺入方法测定VSMC DNA的合成;流式细胞仪分析增殖细胞周期,荧光分光光度计测定VSMC内[Ca2＋]i。结果：PTF以浓度依赖方式抑制PDGF-BB诱导的VSMC增殖、DNA合成,血管平滑肌细胞处于G1/G0期的细胞数增多,而G2/S期的细胞数显著减少（P〈0.01）;能抑制高K＋诱导的VSMC内[Ca2＋]i升高（P〈0.01）。结论：PTF可明显抑制PDGF-BB诱导的VSMC增殖,其作用机制可能与其降低VSMC内高钙浓度[Ca2＋]i有关。     

拉马克啉对内皮素-1诱导的大鼠血管平滑肌细胞增殖及蛋白激酶C表达的作用

目的：探讨拉马克啉（levcromakalim）对内皮素-1（ET-1）诱导的大鼠血管平滑肌细胞增殖及蛋白激酶C（protein kinase C，PKC）表达的影响。方法：体外培养Wistar大鼠主动脉血管平滑肌细胞（vascular smooth muscle cell，VSMCs），用ET-1诱导其增殖，用不同浓度拉马克啉共培养，MTT法评价VSMCs增殖情况，3H—TdR检测DNA合成，流式细胞术检测VSMCs凋亡，逆转录聚合酶链式反应（RT-PCR），Western blot检测PKCamRNA及蛋白表达。结果：拉马克啉对ET-1所致VSMCs增殖有显著抑制作用，随浓度增加，其MTT活性细胞含量和3H—TdR掺入量都明显减少（P〈0．05）；拉马克啉呈剂量依赖性地增加G0／G1期VSMCs（P〈0．05），促使VSMCs凋亡增多（P〈0．05）；拉马克啉抑制VSMCs内PKCamRNA及蛋白表达。结论：拉马克啉抑制ET-1诱导的VSMCs增殖作用，可能的机制是通过下调VSMCs内PKCa表达水平而影响vSMCs增殖和凋亡。     

丹参酮ⅡA对血管紧张肽Ⅱ诱导增殖的 大鼠血管平滑肌细胞中c-fos与c-myc的影响

［摘要］目的探讨丹参酮ⅡA对血管紧张肽Ⅱ（AngⅡ）诱导的大鼠血管平滑肌细胞（VSMC）增殖的影响及其机制。方法分离大鼠胸主动脉中层平滑肌，贴壁法培养平滑肌细胞，建立AngⅡ诱导的VSMC增殖模型；以四甲基偶氮唑蓝（MTT）法观察丹参酮ⅡA对VSMC增殖的影响；流式细胞仪检测药物对细胞周期的影响；应用免疫细胞化学方法观察原癌基因c fos和c myc表达水平的变化。结果成功建立AngⅡ诱导的VSMC增殖模型；随着丹参酮ⅡA浓度增加，VSMC增殖活性呈明显下降趋势（P＜0.05）；流式细胞术结果显示丹参酮ⅡA使VSMC G0/G1期比例升高（P＜0.01），S期比例下降（P＜0.01）;丹参酮ⅡA各组随着浓度的增加，VSMC中c fos和c myc表达水平显著下降（P＜0.05）。结论丹参酮ⅡA具有抑制血管平滑肌细胞增殖的作用，并呈浓度依赖性。其机制可能与丹参酮ⅡA下调VSMC中c fos及c myc表达水平有关。     

黄芪抑制血管平滑肌细胞增殖及其作用机制

目的 观察黄芪(AS)对血管平滑肌细胞(VSMC)增殖的抑制作用，了解黄芪是否通过刺激VSMC产生一氧化氮(nitric oxide，NO)，使细胞周期停滞于G0／G1期，从而抑制平滑肌细胞增殖。方法 [3H]-胸腺嘧啶核苷酸([3H]-TdR)掺入测定VSMCDNA合成，流式细胞仪检测细胞周期情况，硝酸还原酶法测定细胞培养上清中NO水平。结果 黄芪以剂量依赖关系抑制血清诱导的VSMC[3H]-胸腺嘧啶核苷酸掺入，使G0／G1期细胞比例明显增多，S期细胞比例显著减少，黄芪刺激VSMC后，细胞培养上清中NO水平呈剂量依赖上升。结论黄芪能抑制VSMC增殖，使细胞周期停滞于G0／G1期，这一过程可能与黄芪刺激VSMC产生NO有关。     

叶酸抑制同型半胱氨酸诱导大鼠血管平滑肌细胞增殖的研究

目的探讨叶酸抑制同型半胱氨酸致大鼠血管平滑肌细胞增殖的研究。方法培养大鼠胸主动脉血管平滑肌细胞(VSMC),流式细胞仪检测VSMC周期,[3H]TdR参入测定VSMC的DNA合成。结果同型半胱氨酸以剂量依赖关系使VSMC周期中的G0/G1期细胞比例明显减少,S期细胞比例显著增多,增加VSMC的[3H]TdR参入。叶酸可明显抑制同型半胱氨酸诱导的作用。结论同型半胱氨酸能诱导VSMC增殖,叶酸能抑制同型半胱氨酸诱导的VSMC增殖。     

核因子-κB在凝血酶促自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的：研究凝血酶诱导培养的自发性高血压大鼠（SHR）血管平滑肌细胞（VSMCs）的增殖作用，探讨其引起VSMCs增殖与NF—κB途径的关系及可能机制。方法：以0．01、0．1、0．2、0．5、1．0、2．0U／mL的凝血酶与培养的VSMCs共孵育24h和用0．5U／mL凝血酶与VSMCs分别孵育1、2、6、12、24、48h：采用WST-1代谢活性和^3H—TdR掺入率测定VSMCs细胞增殖率；免疫荧光法和蛋白质印迹技术检测基础状态下和0．5U／mL凝血酶干预的VSMCs中NF—κB的细胞内定位及其核中NF—κB的蛋白含量；以已知的AngⅡ和PDGF对VSMCs的增殖作用为阳性对照，研究NF-κB途径与凝血酶诱导的VSMCs增殖关系,100μmol／LPDTC（NF—κB特异性阻断剂）预处理后检测凝血酶引起VSMCs的增殖率。结果：浓度为0．2～2．0U／mL的凝血酶对VSMCs分别有明显的促增殖作用（P〈0．05和P〈0．01）；但浓度为0．5～2．0U／mL的凝血酶作用24h对VSMCs的促增殖作用差异无统计学意义（P〉0．05）。0．5U／mL凝血酶促VSMCs的增殖呈双峰样改变．1h和24h分别达峰值。基础状态下VSMCs的NF-κB主要分布于细胞浆，凝血酶干预后VSMCs的NF—κB主要分布于细胞核。PDTC预处理明显抑制凝血酶促VSMCs增殖的作用（P〈0．01）。结论：浓度0．2～0．5U／mL范围内的凝血酶呈浓度依赖性方式促进培养SHR的VSMCs增殖：而且在时间上呈双峰样改变，提示凝血酶的促增殖作用存在延迟现象。凝血酶能够激活VSMCs的NF—κB，使其从细胞浆转位至细胞核。凝血酶引起VSMCs增殖与NF-κB途径有关联，NF—κB可能是VSMC增殖的细胞内信号转导的共同通路。     

异钩藤碱对缸管平滑肌细胞增殖的影响

目的：研究异钩藤碱对AngⅡ诱导大鼠血管平滑肌细胞（VSMC）增殖的影响，并进一步观察其对细胞增殖周期，原癌基因c-fos、骨桥蛋白（OPN）以及增殖细胞核抗原（PCNA）mRNA表达的作用，旨在探讨异钩藤碱作用的分子机制。方法：采用组织块贴壁法培养Wistar大鼠胸主动脉VSMC，利用细胞计数和MTT比色法检测不同浓度异钩藤碱（10^-7、10^-6、10^-5M）和厄贝沙坦（10^-5M）对AngⅡ／（10^-6M）诱导大鼠VSMC增殖的影响；收集细胞培养上清液测定药物对一氧化氮（NO）含量及一氧化氮合酶（NOS）活性的影响；通过流式细胞仪分析药物刘细胞增殖周期的影响；实时荧光定量PCR检测VSMC中c-fos、OPN、PCNAmRNA表达水平。结果：异钩藤碱和厄贝沙均明显抑制AngⅡ诱导的大鼠VSMC增殖，阻滞细胞由GO／GI期向S期转化，升高上清液中NO含量和NOS活性，随着异钩藤碱浓度的增加，c—fos、OPN、PCNAmRNA表达明显减少。     

D-硫酸多糖抗血管平滑肌细胞增殖作用及其机制

目的：探讨硫酸多糖（DPS）抗增殖作用及其机制,方法;用MTT法评价DPS抗大鼠主动脉平滑肌细胞（VSMC）增殖作用,用流式细胞仪观察DPS对VSMC胞浆内游离Ca^2 的含量、细胞周期和蛋白质含量的影响。结果：DPS在0.001-100mg/L浓度下,对AngⅡ诱导的VSMC增殖均有明显抑制作用,最佳抑制浓度为1mg/L,流式细胞术分析结果表明,DPS在抗增殖的浓度（1mg/L）下能抑制VSMC从G0/G1到S期的转换,阻止VSMC进入G2/M期;减少VSMC蛋白质的合成、DPS能明显抑制胞浆内游离Ca^2 浓度的升高,此作用可被一氧化氮合酶抑制剂（L-NAME）所阻断,提示一氧化氮可能介导了DPS降低胞浆内游离Ca^2 浓度的作用。结论：硫酸多糖DPS可拮抗AngⅡ诱导的VSMC增殖,其作用机理可能与降低胸浆内游离Ca^2 浓度,抑制VSMC的DNA及蛋白质合成有关。     

三七总皂苷对血小板衍生生长因子诱导的血管平滑肌细胞增殖的影响

目的 研究三七总皂苷（TPNS）对血小板衍生生长因子（PDGF）诱导SD大鼠胸主动脉平滑肌细胞（tASMC）增殖的抑制作用及机制.方法 将SD大鼠tASMC进行体外培养,采用四甲基偶氮唑盐比色法检测TPNS对PDGF诱导平滑肌细胞增殖的影响,采用钙荧光指示剂Fura-2/AM负载的平滑肌细胞测定细胞内游离钙浓度.结果 PDGF能诱导血管平滑肌细胞增殖（P〈0.01）,并使细胞内游离钙浓度增高（P〈0.01）,TPNS能抑制该作用（P〈0.01）.结论 TPNS可能通过抑制细胞内游离钙离子浓度的升高来拮抗PDGF诱导的平滑肌细胞增殖.     

三七总皂甙对凝血酶诱血管平滑肌细胞增殖活性的影响

目的探讨三七总皂甙（totalspaoninsofpanaxnotoginseng,tPNS）对凝血酶诱大鼠血管平滑肌细胞（VascularsmoothmUSClecells．vsMcs）的促增殖作用的量效和时效关系。方法采用4～10代的vsMcs的单细胞悬液用于实验。通过MTS法和B—N-乙酰氨基己糖苷酶测定VSMCs细胞增殖率,观察tPNS对凝血酶诱大鼠VSMCs增殖的影响。结果（1）0．5u／ml和1．0u／ml两种浓度的凝血酶均有促VSMCs增殖作用（P〈0．05）。增殖曲线呈双峰样改变（峰值在1h和24h）;（2）不同tPNS浓度（300．0。600．0μg／m1）在24h时对0．5u／ml凝血酶刺激VSMCs都有明显抑制作用,且各浓度之间有显著性差异（P〈0．05）;（3）相对凝血酶组,tPNS（450．0μg／ml）有效地降低了凝血酶（0．5u／m1）诱导VSMCs增殖的1h峰值及24h峰值俨〈0．05）。结论（1）凝血酶促VSMCs的增殖在时间上呈双峰样改变;（2）不同浓度的tPNS对凝血酶诱大鼠血管平滑肌细胞增殖均有抑制作用：（3）tPNS不仅能抑制凝血酶早期快速促VSMCs增殖作用,而且能抑制凝血酶后期促VSMCs增殖作用。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

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.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

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.