LPS对大鼠雪旺氏细胞iNOS表达的影响

目的：探讨内毒素脂多糖（Lipopolysaccharide,LPS）对大鼠雪旺氏细胞（Schwann cells,Scs）诱导型一氧化氮合酶（Inducible nitric-oxide synthase,iNOS）基因表达及一氧化氮（Nitric oxide,NO）生成的影响。方法：用不同浓度（1、10、100μg/ml）和同一浓度不同时间（1、2、4、6小时）的LPS刺激雪旺氏细胞,分别用RT-PCR和亚硝酸盐含量测定观察细胞iNOS mRNA的表达量和细胞培养液中亚硝酸盐的水平,同时用免疫荧光细胞化学染色检测iNOS的细胞定位。结果：用LPS10μg/ml刺激2小时后,iNOS mRNA的表达增加,4小时表达活性最高。细胞上清中的亚硝酸盐含量高峰在6小时。免疫细胞化学证明LPS诱导雪旺氏细胞iNOS的表达定位在胞浆。结论：LPS可在转录水平上诱导雪旺氏细胞iNOS mRNA表达,促进NO的合成,提示雪旺氏细胞在周围神经系统炎症过程中可能发挥免疫调节作用。     

LPS和几种促炎细胞因子促进人血管平滑肌细胞iNOS mRNA的表达

ＬＰＳ、ｒｈｌＬ－１β、ｒｈＩＬ－６、ｒｈＴｈＦ－ａ和ｒｈＩＦＮ－γ单独或两两组合加于培养的人血管平滑肌细胞（ｈＶＳＭＣ）,测定了ｈＶＳＭＣＮＯＳ活性,ｃＧＭＰ含量,ｉＮＯＳｍＲＮＡ表达丰度和培养液中ＮＯ２浓度。ＬＰＳ和细胞因子都能诱导ｈＶＳＭＣ表达ｉＮＯＳ　ｍＲＮＡ,升高ｈＶＳＭＣ　ＮＯＳ活性、ｃＧＭＰ含量和培养液中ＮＯ２浓度;ＴＮＦ－ａ是受试细胞因子中唯一能与另外三种细胞因子分别组合都显示出协同效应的细胞因子;ＬＰＳ和其余三种细胞因子间的两两组合都没有显示出协同效应;本研究结果表明：ＬＰＳ、ｒｈＴＮＦ－α、ｒｈＩＬ－１β、ｒｈＩＬ－６、ｔｈＴｈＦ－α、ｔｈＩＦＮ－γ都能诱导ｈＶＳＭＣ合成ＮＯ;ＴＮＦ－α可能是诱导ｈＶＳＭＣｉＮＯＳ表达的致炎细胞因子中最为重要的。     

雷公藤甲素对LPS诱导的海马炎症过程中COX-2和iNOS表达的影响

选用健康成年雄性SD大鼠,用不同剂量(10μg·kg-1·d-1,25μg·kg-1·d-1,50μg·kg-1·d-1)的雷公藤甲素预处理5d后,立体定位海马注射脂多糖(LPS),采用免疫组织化学染色方法观察海马CA3区环氧化酶2(COX-2)和诱导型一氧化氮合酶(iNOS)的表达变化。结果发现:与注射生理盐水对照组比较,注射LPS可引起海马CA3区的COX-2和iNOS显著表达,而雷公藤甲素(50μg·kg-1·d-1)可明显下调LPS诱导的COX-2和iNOS的表达,其抑制程度与药物剂量呈正相关。本实验结果提示雷公藤甲素对中枢神经系统中LPS诱导的COX-2和iNOS的表达有明显的抑制作用。     

槐定碱对LPS诱导的RAW264.7巨噬细胞CD14、P38、iNOS表达的影响

目的 探讨槐定碱不同给药方式对内毒素诱导的小鼠RAW264.7巨噬细胞表达CD14、p38 MAPK及i NOS的影响及意义。方法 采用LPS诱导的RAW264.7巨噬细胞建立细胞炎症反应模型;实验细胞分为4组(n=6):空白对照组、LPS组、槐定碱预处理组、槐定碱预混合组;分别利用反转录聚合酶链反应(RT-PCR)和Western blot技术检测RAW264.7巨噬细胞CD14、p38、i NOS m RNA表达量及蛋白表达量。结果 槐定碱2种给药方式(预处理及预混合)均可显著下调LPS诱导的RAW264.7巨噬细胞p38、i NOS m RNA及CD14、p-p38、i NOS蛋白表达;槐定碱与LPS预混合可下调LPS诱导的RAW264.7巨噬细胞CD14 m RNA表达,而槐定碱预处理对CD14 m RNA表达无明显影响。结论 槐定碱可能通过调控CD14、p38、i NOS表达与活化发挥抗内毒素效应。     

ERK信号转导途径在LPS诱导的人类内皮细胞iNOS表达中的作用

目的主要研究 p44/42MAPK(ERK1/2或ERK)信号途径在LPS诱导的人内皮细胞 -ECV304iNOS表达和NO产生中的作用。方法用Griess法检测细胞培养液中NO水平 ,分别用免疫荧光法和RT -PCR检测细胞iNOS蛋白和mRNA表达 ,采用免疫激酶沉淀以Westernblot检测细胞中ERK激酶的活性。结果LPS可显著激活人类内皮细胞 -ECV304中的ERK信号途径 ,其激活高峰在LPS刺激后15～20min ,45min后活性逐渐下降。用ERK的特异性抑制剂PD98059处理细胞后可以显著抑制LPS诱导人内皮细胞ERK的活性。与对照组相比 ,LPS刺激后的ECV304细胞iNOS蛋白和mRNA的表达明显增加 ,培养基中NO水平也显著升高 ,用PD98059预处理细胞后 ,可显著抑制细胞iNOS表达和NO和产生。研究结果表明ERK信号途径参与了LPS介导的人内皮细胞iNOS表达和NO产生。结论通过阻断信号转导通路来减少iNOS及其它细胞因子的产生 ,为败血症休克的防治提供了一条新的思路     

纳米银联合放疗对大鼠胶质瘤内iNOS表达和NO水平的影响

目的:研究局部使用纳米银联合放疗对大鼠胶质瘤内诱导型一氧化氮合酶(iNOS)表达和一氧化氮(NO)含量的影响。方法:将C6细胞接种于大鼠右侧尾状核,建模后第8 d,将荷瘤鼠随机分成放疗对照组和纳米银联合放疗组,分别立体定向注入等体积的去离子水及20μg的纳米银,并行单次电离辐射。观察放疗后瘤组织形态学变化,免疫组织化学法检测iNOS蛋白表达,硝酸还原酶法测定NO含量。结果:瘤组织放疗后细胞排列紊乱、密度减少。放疗后6 h胶质瘤内iNOS即有表达,随着时间的延长,iNOS表达进一步增强。放疗后6 h和24h,纳米银联合放疗组iNOS活性均较放疗对照组增高,差异有统计学意义(P<0.01或P<0.001);NO含量的变化与iNOS变化规律基本一致。结论:纳米银联合放疗能够增强iNOS表达,促进NO生成,瘤组织iNOS表达和NO含量改变可能在纳米银联合放疗杀伤胶质瘤细胞的过程中发挥一定的作用。     

小鼠sST2基因真核表达及其对LPS活化RAW264.7巨噬细胞的调节作用

目的:克隆并构建含小鼠sST2和人IgG Fc 融合基因的真核表达质粒, 初步探讨其表达产物sST2-Fc融合蛋白对LPS刺激小鼠巨噬细胞生物学活性的影响.方法:借助RT-PCR技术, 从小鼠脾脏总RNA中扩增全长sST2 cDNA, 构建sST2与hIgG Fc段融合基因的真核表达载体(psST2-Fc).应用脂质体将重组质粒psST2-Fc转染CHO细胞, 经G418筛选, 获得稳定表达sST2-Fc融合蛋白的CHO细胞株.细胞培养上清经蛋白A亲和层析纯化后, 采用Western blot进行鉴定, 应用FACS检测sST2-Fc与RAW264.7巨噬细胞表面相应受体结合情况;ELISA法检测sST2-Fc对LPS刺激RAW264.7巨噬细胞分泌TNF-α和IL-6的影响.结果:测序证实克隆和构建的小鼠sST2-Fc cDNA阅读框序列正确, Western blot 证实sST2-Fc融合蛋白的表达, sST2-Fc抑制LPS诱导巨噬细胞分泌的炎性细胞因子TNF-α和IL-6.结论:成功构建sST2-Fc融合基因并获得稳定表达, sST2-Fc通过与其特异性受体结合可抑制巨噬细胞介导的炎性反应.     

NF—KB“decoy”寡核苷酸对LPS诱导巨噬细胞产生NO及活性氧的影响

目的：探讨NF—KB“decoy”寡核苷酸(0DNs)对脂多糖(LPS)诱导小鼠巨噬细胞株J774．1产生一氧化氮(NO)及活性氧的影响。方法：通过体外细胞培养技术，观察转染NF—KB“decoy”ODNs对LPS刺激巨噬细胞产生NO和活性氧(ROS)的影响，以及诱导型一氧化氮合酶(iNOS)活力的变化。结果：在LPS刺激的巨噬细胞中转染NF—kB“decoy”ODNs减少NO和ROS的产生，降低iNOS的活力。结论：NF—kB“decoy”ODNs可以对抗LPS诱导巨噬细胞产生的ROS和NO，可能与其特异性竞争抑制活化NF—kB结合位点有关。     

参附注射液对肝脏缺血再灌注大鼠iNOS和NO水平的影响

目的探讨参附注射液(SF)对大鼠肝脏缺血再灌注损伤肝组织中诱导型一氧化氮合酶(iNOS)和血清一氧化氮(NO)的影响。方法32只Wistar大鼠随机分为参附实验组(SF组)和肝缺血再灌注组(IR组)。SF组腹腔注射参附注射液(10ml·kg-1),IR组大鼠给予相同剂量的生理盐水。两组均采用Pringle's法阻断肝门缺血15min再灌注1h、3h,测定血清谷丙转氨酶(ALT)、谷草转氨酶(AST)以及NO水平,并应用免疫组织化学法测定肝组织中iNOS表达。结果大鼠肝脏缺血15min再灌注1h和3h,SF组血清ALT、AST以及NO水平低于IR组(P<0.05),SF组肝组织iNOS阳性产物平均吸光度值、阳性面积百分率(%)明显低于IR组(P<0.05)。结论参附注射液抑制iNOS的表达,减少过量NO的生成,可能是其对肝脏缺血再灌注损伤的保护作用机制之一。     

NF-κB "decoy"寡核苷酸对LPS诱导巨噬细胞产生NO及活性氧的影响

目的 :探讨NF -κB“decoy”寡核苷酸 (ODNs)对脂多糖 (LPS)诱导小鼠巨噬细胞株J774 1产生一氧化氮 (NO)及活性氧的影响。方法 :通过体外细胞培养技术 ,观察转染NF -κB“decoy”ODNs对LPS刺激巨噬细胞产生NO和活性氧 (ROS)的影响 ,以及诱导型一氧化氮合酶 (iNOS)活力的变化。结果 :在LPS刺激的巨噬细胞中转染NF -κB“decoy”ODNs减少NO和ROS的产生 ,降低iNOS的活力。结论 :NF -κB“decoy”ODNs可以对抗LPS诱导巨噬细胞产生的ROS和NO ,可能与其特异性竞争抑制活化NF -κB结合位点有关。     

CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264.7)

Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL-12 and TNF-alpha. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264.7). Neutralizing antibody against TNF-alpha was not able to inhibit NO or iNOS production from the CpG ODN 1826-activated macrophages, suggesting that although the TNF-alpha was also produced by CpG ODN-activated macrophages, the production of iNOS was not mediated through TNF-alpha. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826-activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF-kappaB translocation, as indicated by the delay of IkappaBalpha degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification.     

三黄泻心汤对LPS诱导的RAW264.7细胞释放炎性细胞因子影响的研究

目的:观察三黄泻心汤对LPS刺激小鼠RAW264.7细胞释放炎性细胞因子的影响。方法:体外培养小鼠RAW264.7细胞,LPS刺激小鼠RAW264.7细胞分泌细胞因子,用不同浓度三黄泻心汤及三黄泻心汤有效组分进行干预,ELISA法检测细胞培养上清液中TNF-α含量。结果:三黄泻心汤及三黄泻心汤有效组分可抑制LPS刺激小鼠RAW264.7细胞释放TNF-α,并且其影响细胞因子释放作用与细胞毒作用无关。结论:三黄泻心汤及其有效组分B2、B3可抑制LPS刺激小鼠RAW264.7细胞释放TNF-α。     

Effects of progesterone and estradiol sex hormones on the release of microparticles by RAW 264.7 macrophages stimulated by Poly(I:C)

Microparticles (MPs) are small membrane-bound vesicles that display proinflammatory and prothrombotic properties. These particles can be released by macrophages stimulated by ligands of the Toll-like receptors (TLRs) in a process that depends on nitric oxide (NO) production. Since sex hormones can modulate macrophage responses, we investigated the effects of progesterone and estradiol on macrophage particle release in vitro, comparing the responses with those induced by the glucocorticoid dexamethasone. As a model system for particle release, RAW 264.7 cells were stimulated in vitro with poly(I:C), a ligand of TLR3. Microparticles were measured by flow cytometry, while NO was measured by the Griess reaction. As the results of these studies showed, progesterone but not estradiol can block particle release by RAW264.7 cells treated with poly(I:C); dexamethasone was also active. Furthermore, while progesterone and dexamethasone inhibited NO production under the same culture conditions, neither agent blocked the production of particles stimulated by the NO donors dipropylenetriamine NONOate {(z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate} and (z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate. Studies using RU486 to assess the role of hormone receptors indicated that while this agent blocked the inhibition of particle and NO production by dexamethasone, it did not affect the inhibition by progesterone. Together, these results indicate that progesterone but not estradiol can inhibit particle release by stimulated macrophages and suggest a mechanism that may contribute to the immunomodulatory effects of this sex hormone.     

需钙蛋白酶抑制剂Ⅰ对LPS攻击的RAW264．7细胞中iκBα表达和细胞因子分泌的影响

目的：探讨需钙蛋白酶抑制剂I（CI-I）对LPS攻击RAW264．7细胞后iκBα表达和细胞因子分泌的影响。方法：用CI—I预处理RAW264．7细胞1h后，再用LPS攻击，分别用Western blot和ELISA检测RAW264．7细胞iκBα蛋白表达和TNF-α、IL-6的分泌。结果：CI—I预处理RAW264．7细胞后，可抑制LPS导致的iκBα表达降低。LPS攻击RAW264．7细胞后4h和8h，TNF—α和IL-6的分泌均增加，CI—I和地塞米松（DEX）可抑制该效应，并具有协例抑制作用。结论：CI—I和DEX可抑制LPS攻击后RAW264．7细胞中iκBα表达和炎性细胞因子分泌，从而有助于减轻细胞损伤。     

姜黄素对LPS刺激RAW264.7细胞PGE_2和IL-10影响

目的:观察姜黄素对脂多糖刺激小鼠RAW264.7细胞炎症因子的影响。方法:体外培养小鼠RAW264.7细胞,脂多糖刺激小鼠RAW264.7细胞分泌细胞因子,用不同浓度姜黄素进行干预,ELISA法检测培养上清液中抗炎细胞因子IL-10和炎症因子PGE2含量。结果:姜黄素可抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进抗炎因子IL-10表达,并呈一定量效关系,其影响细胞因子释放作用与细胞毒作用无关。结论:姜黄素呈浓度依赖性地抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进IL-10表达。     

IL-15 induces IFN-beta and iNOS gene expression, and antiviral activity of murine macrophage RAW 264.7 cells

The effects of interleukine-15 (IL-15) on macrophage activation and antiviral activity have been investigated in this study. We have provided evidence that IL-15 stimulates murine macrophage RAW 264.7 cells to release nitric oxide (NO) and inhibit vaccinia virus (VV) replication in bystander human 293 cells in a dose-dependent manner. The IL-15-induced antiviral activity was partially mediated by NO, as blocking NO production by NO synthase (iNOS) inhibitor NG-monomethyl-L-arginine acetate (L-NMA) partially restored the virus replication. Interferon-gamma (IFN-gamma) was not detectable by ELISA in the cell supernatant of IL-15-activated macrophages or in the co-cultures of macrophages and infected bystander cells. Neutralizing anti-IFN-gamma, anti-IFN-gamma receptor R2, anti-TNF-alpha, or anti-IL-12 antibodies had no effect on NO production or antiviral activity. In contrast, neutralizing anti-IFN-alpha/beta antibody completely restored the VV replication and reduced the NO level to one third of that in the control. Elevated mRNA levels of IFN-beta and iNOS genes were detected in IL-15-activated RAW 264.7 cells by RT-PCR. Our data suggest that IL-15 is capable of inducing IFN-beta, which could participate in NO-mediated antiviral effect.     

Effect of macrophage colony stimulating factor overexpression on oxidative injury/resistance of RAW264.7 cells

Oxidative injury to monocytes/macrophages is considered one of the key factors in atherogenesis. Macrophage colony stimulating factor (M-CSF) also plays an important role in the stages of atherosclerosis. Some researchers showed that M-CSF accelerated pathological changes in the early stages of atherosclerosis. However, other reports suggested that exogenous M-CSF could prevent the progress of atherosclerosis. To further investigate the role of M-CSF in atherogenesis and to elucidate the effect of M-CSF on the oxidative injury to monocytes/macrophages, RAW264.7 cell lines overexpressing M-CSF were established by applying the lipofectin transfection method. The oxidative injurious effect of tert-butylhydroperoxide on the established cell lines was investigated. Two M-CSF-transfected RAW264.7 cell lines secreted large amounts of M-CSF. Compared with the non-transfected RAW264.7 cells, M-CSF-overexpressing RAW264.7 cells were more vulnerable to oxidative injury. We conclude that M-CSF could aggravate the oxidative injury due to macrophages in some situations. Received: 21 August 2002 / Accepted: 12 March 2003 Correspondence to Z.-J. Pang     

壮药赪桐乙酸乙酯部位及流份对LPS诱导的RAW264.7细胞的抗炎作用

目的 筛选赪桐乙酸乙酯部位及不同洗脱梯度二氯甲烷-甲醇部位对炎症反应的抑制作用最强的流份。 方法 使用MTT法确定壮药赪桐乙酸乙酯部位及不同洗脱梯度二氯甲烷-甲醇部位对RAW264.7细胞安全给药浓度范围,通过ELISA法测定赪桐乙酸乙酯部位及不同洗脱梯度二氯甲烷-甲醇部位对LPS诱导RAW264.7细胞分泌NO、TNF-ａ、IL-12、IL-6、IL-1β含量,筛选对炎症反应的抑制作用最强的流份。 结果 赪桐乙酸乙酯部位及流份在0.06~2.0 mg/ml浓度范围内,赪桐乙酸乙酯部位及流份对细胞活力的抑制作用逐渐增强,浓度在0.5 mg/ml以上具有明显的细胞毒性,在0.5 mg/ml以下对细胞活力具有增强作用。二氯甲烷-甲醇洗脱部位高剂量能抑制炎症因子IL-12、IL-6、TNF-ａ、IL-1β释放,对NO的分泌没有抑制作用。 结论 赪桐乙酸乙酯部位及不同洗脱梯度的二氯甲烷-甲醇部位抗炎机制是通过抑制NO、TNF-ａ、IL-12、IL-6、IL-1β炎症因子的分泌,二氯甲烷-甲醇（50:1）洗脱部位和二氯甲烷-甲醇（30:1）洗脱部位的抗炎能力较强。     

CCK-8降低LPS诱导的小鼠单核细胞RAW264.7 CHOP/caspase-11/caspase-1的表达

目的探讨CCK-8对LPS激活RAW264.7细胞CHOP/caspase-11/caspase-1信号通路的影响。方法用LPS和(或)CCK-8和(或)CCK-8的1受体阻滞剂孵育RAW 264.7细胞,用Western blot法检测CHOP、caspase-11蛋白表达,用试剂盒检测caspase-1活性,用ELISA技术检测细胞培养上清中IL-1β水平。结果与对照组相比,LPS组CHOP、caspase-11表达水平均显著升高(P0.05),给予CCK-8可有效抑制LPS诱导的CHOP、caspase-11表达升高(P0.05),而预先给予CCK-8的1受体阻滞剂可明显抑制CCK-8的作用。caspase-1活性变化、IL-1β含量变化趋势均与CHOP、caspase-11表达水平变化趋势一致(P0.05)。结论 CCK-8通过受体1抑制CHOP/caspase-11/caspase-1表达,减少LPS诱导IL-1β的生成,发挥抗炎作用。     

需钙蛋白酶抑制剂I对LPS攻击的RAW264.7细胞中iκBα表达和细胞因子分泌的影响

目的:探讨需钙蛋白酶抑制剂I(CI-I)对LPS攻击RAW264.7细胞后iκBα表达和细胞因子分泌的影响。方法:用CI-I预处理RAW264.7细胞1h后,再用LPS攻击,分别用Westernblot和ELISA检测RAW264.7细胞iκBα蛋白表达和TNF-α、IL-6的分泌。结果:CI-I预处理RAW264.7细胞后,可抑制LPS导致的iκBα表达降低。LPS攻击RAW264.7细胞后4h和8h,TNF-α和IL-6的分泌均增加,CI-I和地塞米松(DEX)可抑制该效应,并具有协同抑制作用。结论:CI-I和DEX可抑制LPS攻击后RAW264.7细胞中iκBα表达和炎性细胞因子分泌,从而有助于减轻细胞损伤。