齐墩果酸抑制肿瘤坏死因子-α诱导的成纤维细胞样滑膜细胞炎症因子表达及其机制研究

探讨齐墩果酸(Oleanolic acid,OA)对肿瘤坏死因子-α(TNF-α)诱导成纤维细胞样滑膜细胞的炎症因子表达的影响及其机制。首先复苏培养人成纤维细胞样滑膜细胞(FLS),通过RT-PCR检测细胞IL-6及IL-1βmRNA表达,采用Western blot方法检测p38MAPK及NF-κB蛋白表达变化,通过ELISA法检测细胞上清液中IL-6及IL-1β浓度。与对照组比较,TNF-α明显诱导FLS细胞IL-6及IL-1βmRNA的表达及上清液中IL-6及IL-1β的分泌(P0.05),同时磷酸化p38蛋白和核NF-κB明显增加(P0.05),且p38MAPK阻断剂SB203580能抑制TNF-α诱导的核NF-κB增加。OA呈浓度依赖性抑制TNF-α诱导的FLS细胞p38蛋白磷酸化和核NF-κB增加(P0.05)。且OA、p38MAPK通路抑制剂SB203580或NF-κB阻断剂BAY 11-7082均能抑制TNF-α诱导的IL-6及IL-1β分泌增加(P0.05)。综上所述,OA能抑制TNF-α诱导的FLS细胞炎症因子IL-6及IL-1β的产生,其机制可能与抑制p38MAPK/NF-κB信号通路有关。     

黄芩苷对脂多糖诱导的巨噬细胞NF-κB活化和炎症因子表达的影响

目的:研究黄芩苷对脂多糖(LPS)诱导小鼠巨噬细胞核因子κB(NF-κB)及肿瘤坏死因子α(TNF-α)、白介素6(IL-6)表达的影响.方法:分别用LPS(终浓度1μgomL-1)和LPs+黄芩苷(终浓度10,50,100μmol moloL-1)处理生长良好的小鼠巨噬细胞RAW264.7,用RT-PCR法和Elisa法检测细胞及其上清液中TNF-α、IL-6 mRNA和蛋白的表达变化,用Western Blot法检测细胞核内NF-κB p65蛋白含量变化.结果:LPS刺激RAW264.7细胞可导致NF-κB激活,上调TNF-α、IL-6表达;黄芩苷预处理能降低LPS诱导的NF-κB出活化和TNF-α、IL-6表达.结论:黄芩苷可通过抑制NF-κB活化,下调LPS诱导的巨噬细胞TNF-α、IL-6的生成,发挥抗炎作用.这可能是其抗动脉粥样硬化的作用机制之一.     

胰岛素在巨噬细胞泡沫化过程中对Toll样受体4表达的影响及其机制

目的:观察胰岛素对巨噬细胞破泡沫化过程中Toll样受体4(TLR4)表达及IL-6、TNF-α分泌的影响.方法:采用体外培养小鼠巨噬细胞系RAW264.7,氧化低密度脂蛋白(ox-LDL)诱导建立泡沫细胞模型,分为对照组、ox-LDL组、用胰岛素组、PI3K-AKT抑制剂组.油红O染色观察泡沫细胞模型的建立,取细胞上清用ELISA法检测白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平;流式细胞术检测膜蛋白TLR4表达量,Western-blot检测TLR4、核因子-κB (NF-κB)的表达水平.结果:与对照组相比,ox-LDL处理过的巨噬细胞可向泡沫细胞转换,同时TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著增加(P＜0.05);而使用胰岛素干预后ox-LDL的作用显著减弱,TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著降低(P ＜0.05 vs ox-LDLgroup);而使用PI3K-AKT抑制剂干预后,抑制剂显著降低胰岛素的作用,TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著升高(P ＜0.05 vs ox-LDL+ insulin).结论:ox-LDL可诱导巨噬细胞向泡沫细胞转化,同时上调TLR4及NF-κB蛋白表达,增加炎性因子分泌,促进了AS进程,而胰岛素可使ox-LDL的作用显减弱,减少TLR4、NF-κB蛋白表达及炎性因子分泌,从而减轻AS进程,其机制可能与胰岛素通过PI3K-AKT抑制TLR4-NF-κB通路有关.     

大黄素抑制LPS诱发的结肠癌转移的研究

研究大黄素对炎症介导的结肠癌细胞转移的抑制作用。采用MTT法确定大黄素有效作用浓度。细胞划痕、Transwell、基质胶实验检测大黄素对LPS诱发的结肠癌细胞SW480的迁移、侵袭能力的影响。ELISA实验检测经药物处理后细胞培养基中炎性因子的变化;蛋白质免疫印迹检测LPS单独处理和LPS、大黄素共处理结肠癌细胞后,胞内EMT标志蛋白及炎症信号通路相关蛋白的表达变化;建立小鼠肺转移模型评价大黄素抑制LPS诱发的结肠癌转移的能力。结果发现,大黄素能有效抑制LPS诱发的结肠癌细胞SW480增殖、迁移和侵袭;大黄素与LPS共处理和LPS单独处理结肠癌细胞相比,培养基上清中炎性因子IL-1β、TNF-α、IL-6含量下降;此外,蛋白质免疫印迹结果显示大黄素与LPS共处理较LPS单独处理,EMT过程受到抑制,NF-κB、TLR4表达下调;肺转移模型实验显示大黄素能抑制LPS诱发的结肠癌细胞的肺转移。实验结果表明,大黄素通过抑制LPS诱发炎症因子的释放和炎性信号通路激活,进而抑制结肠癌细胞转移。     

红景天苷对脂多糖诱导小鼠巨噬细胞炎性介质分泌的影响

本文旨在探讨红景天苷(salidroside,Sal)对脂多糖(lipopolysaccharide,LPS)诱导小鼠巨噬细胞系J774.1炎性活化的影响及其可能机制。J774.1细胞分为PBS对照组、LPS(0.5μg/m L)刺激组和不同剂量(5、25、125μg/m L)Sal预处理+LPS组。CCK-8比色法检测细胞活性,ELISA测定培养上清中TNF-α、MCP-1和MIP-2含量,硝酸还原酶法测定上清中NO含量,RT-PCR检测细胞i NOS m RNA表达,Western blot检测胞浆i NOS蛋白和胞浆与胞核NF-κB/p65蛋白表达,Trans AMTM NF-κB/p65活性检测试剂盒测定NF-κB/p65 DNA结合活性。结果显示,0.5μg/m L LPS以及不同剂量(5、25、125μg/m L)Sal处理细胞12 h对J774.1细胞活力无影响;与LPS刺激组比较,LPS刺激前Sal预处理J774.1细胞,培养上清中TNF-α、MCP-1、MIP-2和NO含量呈剂量依赖性降低(P0.05),细胞i NOS m RNA和蛋白表达水平下调(P0.05),胞核NF-κB/p65蛋白表达降低(P0.05)而胞浆NF-κB/p65蛋白相应增加(P0.05),且NF-κB/p65 DNA结合活性呈剂量依赖性降低(P0.05)。以上结果提示,Sal预处理能够降低LPS诱导的巨噬细胞炎性活化,其机制可能通过干扰LPS/TLR4/NF-κB信号通路,从而降低炎性介质及细胞因子的过度表达和分泌。     

乳酸调控大鼠肠黏膜微血管内皮细胞的NF-κB信号通路

目的探讨内毒素（LPS）刺激大鼠肠黏膜微血管内皮细胞（RIMMVECs）后,乳酸（LA）调控NF-κB信号通路中磷酸化IκBα和NF-κB p65蛋白表达情况,肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）mRNA表达情况,阐明乳酸发挥作用的最佳时间及其调控NF-κB信号通路的部位。方法提取RIMMVECs总蛋白和总RNA,用Western blotting检测NF-κB p65、IκBα及p-IκBα蛋白表达水平,用real-time PCR对TNF-α和IL-6 mRNA进行定量检测。结果乳酸能降低LPS诱导RIMMVECs分泌的TNF-α和IL-6 mRNA表达水平,并分别于24 h和3 h下调效果最明显;乳酸能抑制IκBα磷酸化及NF-κB转录活性,并于4～8 h达到最佳效果;乳酸发挥作用部位是抑制信号通路中IκBα磷酸化。结论乳酸通过抑制IκBα磷酸化而阻断NF-κB的激活,抑制下游炎性因子表达,进而发挥出很好的预防炎症效果。     

皮层星形胶质细胞Toll样受体4在炎症和β淀粉样蛋白形成中的作用（英文）

为了探讨星形胶质细胞在炎症和β淀粉样蛋白(amyloidβ-protein,Aβ)形成中的作用和可能的分子机制,本研究通过LPS刺激体外培养大鼠皮层星形胶质细胞,首先采用实时PCR和Western blot方法分别检测Toll样受体4(Toll-like receptor 4,TLR4)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白细胞介素-1β(interleukin 1β,IL-1β)、β淀粉样前体蛋白(β-amyloid precursor protein,β-APP)和β-位点APP剪切酶1(β-site APP clearing enzyme 1,BACE1)m RNA及TLR4、NF-κB/P65蛋白水平,而后用免疫荧光法进一步证明NF-κB/P65的核易位,ELISA法测定培养上清中TNF-α、IL-1β和Aβ含量。结果显示,这些指标在LPS刺激后均不同程度地上调。然而,如果预先用TLR4抗体处理,与仅用LPS刺激组相比,LPS对NF-κB/P65核易位及培养上清中TNF-α、IL-1β和Aβ含量的刺激作用显著减弱或消失。结果表明,星形胶质细胞TLR4可能通过TLR4/NF-κB信号通路在炎症和Aβ的形成中发挥重要的作用。     

银杏叶提取物对内毒素诱导巨噬细胞核因子-κB活化及炎性细胞因子表达的调节

探讨银杏叶提取物（EGb761）对内毒素（LPS）诱导RAW264．7细胞核因子-κB（NF-κB）活化及炎性细胞因子基因表达的调节,为银杏叶提取物的临床运用提供理论依据．分别用LPS或EGb761＋LPS处理体外培养的小鼠巨噬细胞系RAW264．7细胞,采用蛋白质印迹分析检测细胞中NF-κB活性,用逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附法（ELISA）检测细胞中TNF-α、IL-1β、IL-6 mRNA和蛋白的表达．研究结果表明LPS组NF-κB活性和TNF-α、IL-1β、IL-6含量在刺激后2～12h明显高于正常对照组,而EGb761＋LPS组NF-κB活性和TNF-α、IL-1β、IL-6含量均显著低于LPS组．结果提示LPS可诱导RAW264．7细胞NF-κB活化,导致TNF-α、IL-1β、IL-6基因表达增强,而EGb761能抑制NF-κB活化而调节TNF-α、IL-1β、IL-6基因的表达．     

脂多糖诱导成骨细胞中CHI3L1表达的分子机制研究

慢性骨髓炎因其病程漫长、易出现并发症以及复发率高成为临床上棘手的难题,其主要致病原因是金黄色葡萄球菌等革兰氏阴性菌感染．脂多糖(LPS)是革兰氏阴性细菌细胞壁的重要成分,用LPS在体外刺激骨组织相关细胞在一定程度上可以模拟骨髓炎患者的病理特征．实时荧光定量PCR和Western blot等试验结果表明,在骨髓炎患者的骨组织和LPS刺激的成骨细胞中,几丁质酶家族成员CHI3L1的表达均有明显升高．核因子κB (NF-κB) 萤光素酶报告载体检测结果显示,LPS能诱导细胞的NF-κB活化,NF-κB活化抑制剂Bay11-7082能抑制LPS诱导的CHI3L1表达升高．用抗肿瘤坏死因子α(TNF-α)的抗体预处理细胞,或采用siRNA干扰的方法抑制TNF-α受体的表达,都能明显抑制LPS诱导的CHI3L1表达上调．同时,NF-κB活化抑制剂Bay11-7082预处理细胞能抑制LPS对TNF-α表达的诱导作用．结果提示,LPS通过激活NF-κB诱导TNF-α分泌上调,刺激CHI3L1表达．提出骨髓炎及脂多糖刺激条件下CHI3L1表达上调,并在细胞水平上初步探讨了脂多糖诱导CHI3L1表达的分子机制．     

Rig-I对LPS诱导的巨噬细胞增殖、凋亡及功能的作用研究

该研究旨在探讨维甲酸诱导基因I(retinoic acid-inducible gene I,Rig-I)对脂多糖(lipopolysaccharide,LPS)诱导的巨噬细胞增殖、凋亡和细胞因子分泌等生物学功能的影响及其作用机制。以不同剂量的LPS刺激Rig-I基因沉默和过表达的小鼠巨噬细胞系Raw264.7细胞,采用CCK-8和流式细胞术检测细胞的生长状态,采用q PCR方法检测细胞因子TNF-α、IL-10、IL-1和IL-6相关基因的表达水平,并采用Western blot方法检测LPS诱导的TLR4信号通路的相关蛋白表达水平。CCK-8和流式细胞术结果显示,Rig-I促进巨噬细胞的增殖并抑制LPS诱导的细胞凋亡;q PCR结果表明,Rig-I促进巨噬细胞中TNF-α、IL-10、IL-1和IL-6基因的表达。进一步的实验证实,Rig-I通过激活AKT及其下游蛋白p-38、NF-κB和Bcl-x L等抑制细胞凋亡并促进细胞因子的表达。该研究首次证实了Rig-I可通过AKT信号通路对LPS诱导的巨噬细胞增殖、凋亡及功能进行调节。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Coiled-coil nanomechanics and uncoiling and unfolding of the superhelix and alpha-helices of myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

放牧对牧草光合作用、呼吸作用和氮、碳吸收与转运的影响

研究放牧对草地植物生理活动的影响,对于揭示草地放牧演替的生理机制有重要意义.大量研究表明,家畜放牧对牧草光合作用、呼吸作用以及C和N吸收与转运的影响,可以分为生理伤害和生理恢复2个阶段.放牧通过改变草地冠层结构影响牧草光合作用,净光合作用速率短期内迅速下降,随着叶面积指数增加又逐渐上升,呼吸作用有相似的变化趋势.牧草放牧后再生长所需的C和N最初主要来自根系和留茬中的贮藏物质,此后随着牧草生长恢复逐渐由同化作用供给,C代谢与土壤N水平负相关.放牧后牧草生理活动变化与牧草遗传特性、种间竞争、家畜放牧特征、非生物环境等因素密切相关.     

Synthesis of hapten and conjugates of coumestrol and development of immunoassay

3-O-Carboxymethylcoumestrol was prepared as the hapten for immunoassay by a partial alkylation of coumestrol with ethyl chloroacetate in acetone alkalized with potassium carbonate. 3-O-Ethoxycarbonylmethylcoumestrol was separated by column chromatography and finally was hydrolyzed with formic acid. 1H and 13C NMR data (APT, COSY, HMQC, and HMBC) revealed that the reaction was regioselective, as 3-O-ethoxycarboxymethylcoumestrol was the only monosubstituted derivative. The hapten was then conjugated to bovine serum albumin and used for immunization of rabbits. A radioimmunoassay (RIA) system was established based on the polyclonal antiserum and a 125I-labeled hapten-tyrosine methyl ester conjugate as the radioligand. Parameters of the RIA: sensitivity: 12 pg per tube, 50% intercept: 140 pg per tube, working range: 20-4000 pg per tube. The cross-reactivity of a panel isoflavonoid and lignan phytoestrogens was either negligible (e.g. formononetin 0.07%; biochanin A 0.06%) or not detectable at all. The major immunoreactive peak in HPLC fractions from an alfalfa extract had the same retention time as coumestrol standard and represented 94.8% of the signal. The remaining 5.2% of immunoreactivity was distributed between five minor peaks. We conclude that after the validation for particular matrices, the method will be a useful tool for analysis of coumestrol, especially in low volume and low concentration samples.