应用RAW264．7-L929细胞系建立一种新型抑制性ODN的筛选方法

目的:为研究拮抗TNF-α的新型免疫抑制性ODN建立一种快速的筛选方法.方法:以CpG ODN 1826作为刺激剂,将其作用于小鼠巨噬细胞RAW264.7细胞,6 h后收集上清,作用于L929细胞,利用L929是TNF-α敏感细胞的原理,检测TNF-α活性.结果:筛选条件确定为CpG ODN 1826终浓度为20 mg/L,RAW264.7上清液1/80倍稀释.放线菌素D浓度为1 mg/L,L929细胞数在2×104个/孔,反应时间为18 h.应用此条件成功筛选出自行设计的抑制性ODN-IM003.结论:建立的实验体系,能很好的区分自行没计的ODN抑制CpG ODN 1826诱导的RAW264.7细胞的TNF-α的生物学活性.     

双氢青蒿素对CpG ODN诱导小鼠RAW264.7细胞释放细胞因子的影响

目的：观察双氢青蒿素对CpG ODN诱导小鼠RAW264．7细胞释放细胞因子的影响。方法：体外培养小鼠RAW264．7细胞．用CpG ODN刺激小鼠RAW264．7细胞分泌TNF-α、IL-6,用不同浓度双氢青蒿素进行拮抗,ELISA法检测培养上清中促炎细胞因子的含量．结果：双氢青蒿素可抑制CpG ODN刺激小鼠RAW264．7细胞释放TNF—α和IL-6,并呈一定的量效关系,其中对IL-6最大抑制率可达到66．5％,而对TNF—α释放的最大抑制率仅为7．2％,其影响细胞因子释放的作用与细胞毒作用无关。结论：双氢青篙素呈浓度依赖性地抑制CpG ODN刺激小鼠RAW264．7细胞释放TNF—α和IL-6,但对TNF—α的影响较小。     

血管紧张素Ⅱ1型受体在脂多糖诱导RAW264.7巨噬细胞促炎性细胞因子产生中的作用

目的 探讨血管紧张素Ⅱ1型受体在脂多糖（LPS）诱导巨噬细胞促炎性细胞因子产生中的作用和机制.方法 按照随机数字表法将小鼠单核巨噬细胞株（RAW264.7）细胞分为空白对照组、ZD7155组、LPS组和LPS ＋ZD7155组.酶联免疫吸附试验法（ELISA）测定各组细胞培养上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的含量,逆转录聚合酶链式反应（RT-PCR）检测RAW264.7细胞内TNF-α和IL-1β mRNA表达,凝胶电泳迁移检测法（EMSA）检测RAW264.7细胞内核因子-κB（NF-κB）和活化蛋白-1（AP-1）活性的变化.结果 和空白对照组相比,ZD7155组培养上清液中的TNF-α和IL-1β含量差异没有统计学意义（P＞0.05）,但是LPS组细胞培养上清液中的TNF-α和IL-1β含量均显著高于空白对照组（均P〈0.01）和ZD7155组（均P〈0.05）.LPS组细胞内的TNF-α和IL-1β mRNA表达是空白对照组的2.19倍（P〈0.01）和1.77倍（P〈0.01）,细胞内NF-κB和AP-1活性是空白对照组的1.43倍（P〈0.01）和1.90倍（P〈0.01）.而LPS＋ZD7155组上清液中的TNF-α和IL-1β含量较LPS组下降（均P〈0.05）,细胞内TNF-α和IL-1β mRNA表达较LPS组下降了34.7%（P〈0.01）和49.72%（P〈0.01）,同时细胞内NF-κB活性和AP-1活性较LPS组下降了46.15%（P〈0.05）和48.42%（P〈0.05）.结论 血管紧张素Ⅱ1型受体通过活化转录因子NF-κB和AP-1,参与了LPS诱导巨噬细胞促炎性细胞因子TNF-α和IL-1β的产生和释放.     

云芝多糖B抑制ox-LDL诱导巨噬细胞株单核细胞趋化蛋白-1mRNA表达的调控机制

目的：探讨云芝多糖（CVPS）-CVPS-B对氧化修饰低密度脂蛋白（ox-LDL）诱导的巨噬细胞(RAW264.7)单核细胞趋化蛋白-1（MCP-1）mRNA表达的影响，及对核因子-κB（NF-κB）和细胞内谷胱甘肽（GSH）的调控作用。方法:应用反转录聚合酶链反应（RT-PCR）测定RAW264.7细胞MCP-1 mRNA的表达；凝胶滞留法（EMSA）测定NF-κB的结合活性；荧光分光光度法测定细胞内GSH的活性。结果:CVPS-B呈剂量依赖性抑制ox-LDL诱导的RAW264.7细胞MCP-1 mRNA的表达。应用GSH 特异性消耗剂buthionine-［S，R］-sulfoximine （BSO），ox-LDL不能诱导RAW264.7细胞MCP-1 mRNA的表达。CVPS-B抑制ox-LDL诱导RAW264.7细胞内GSH活性下降。CVPS-B呈剂量依赖性抑制 ox-LDL诱导的RAW264.7细胞NF-κB活性；在完全消耗GSH的RAW264.7细胞，ox-LDL不能诱导NF-κB的活性。结论:CVPS-B可抑制ox-LDL诱导RAW264.7细胞MCP-1 mRNA的表达；其抑制作用可能通过GSH/NF-κB的调控。     

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。     

CpG序列的免疫激活机理及应用

未甲基化CpG的寡核苷酸片段（CpG oligonucleotides,CpG ODN）是细菌DNA的免疫刺激作用的最小作用单位．广泛存在于细菌DNA中。CpG是以未甲基化的胞嘧啶鸟嘌呤二核苷酸（CpG）为核心的回文序列,5’端为2个嘌呤,3’端为2个嘧啶,即5’-PurPur-CG-PyrPyr-3’。该序列在原核生物、病毒基因组中出现的频率为1／16,通常是非甲基化的;而在脊椎动物的出现频率则大大的降低,且通常甲基化。脊椎动物正是通过这种差异来识别自体和异体CpG;而哺乳动物细胞则是通过TLR9（Toll-like receptor 9,TLR9）识别细菌未甲基化CpG基元。触发一系列机体防御机制,包括补体激活、吞噬作用和致炎细胞因子基因的表达。目前已知具有较强免疫刺激作用的CpG DNA序列包括5’-TCCATGACGTTCCTGACGTT-3’等。     

增强CpG免疫刺激作用途径的研究进展

<正>CpG基序(CpG motifs)是指含有非甲基化的胞嘧啶(C)和鸟嘌呤(G)核苷酸核心的寡聚脱氧核苷酸(ODN)序列,又称为免疫刺激序列,一般为6个寡聚核苷酸。1995年,Krieg等[1]研究发现非甲基化的CpG二寡聚核苷酸是病原体DNA免疫刺激活性的结构基础,自此,对CpG基序的各种研究得以展开,包括CpG基序免疫刺激机制、CpG基序个数及结构对免疫刺激活性影响、CpG寡聚脱氧核苷酸作为佐剂使用等。含有非甲基化CpG二核苷酸的寡聚脱氧核苷酸作为一种免疫增强剂,在体内和体外均表现免疫刺激作用,可以引起先天性免疫反应。CpG在体内能引起明显且多样化     

基于 Caspase-1 细胞焦亡信号通路探讨栀子苷对脂多糖诱导 RAW264.7 细胞炎症的抑制作用

目的：探讨栀子苷对脂多糖（LPS）诱导的小鼠巨噬细胞RAW264.7 Caspase-1细胞焦亡经典信号通路的干预作用。方法：LPS诱导RAW264.7细胞构建炎症模型,以地塞米松为阳性对照,20、40、80μmol/L栀子苷干预细胞24 h。乳酸脱氢酶（LDH）细胞释放法检测细胞LDH释放;碘化丙啶（PI）荧光染色观察细胞存活情况;检测细胞上清中炎症因子IL-18、IL-1β、TNF-α和IL-6分泌水平;Western blot检测NLRP3、Caspase-1、GSDMD、IL-18和IL-1β表达。结果：与正常对照组相比,模型组RAW264.7细胞中LDH释放量明显升高（P<0.01）,细胞形态学观察到有大量细胞被PI染色,细胞上清中IL-1β、IL-18、TNF-α和IL-6分泌水平显著升高（P<0.01）;NLRP3、Caspase-1、GSDMD、IL-18和IL-1β蛋白表达显著升高（P<0.01）。与模型组相比,栀子苷各组和地塞米松组LDH释放减少（P<0.01）,有效抑制PI染色,细胞上清中炎症因子IL-1β、IL-18、TNF-α和IL-6...     

雷帕霉素抑制LPS诱导的RAW264.7细胞表达和释放HMGB1的机制

目的：探讨雷帕霉素抑制LPS诱导小鼠RAW264.7细胞表达和释放HMGB1的作用机制。方法:传代培养的小鼠RAW264.7细胞分5组接种于6孔板，即仅加培养液的对照组；加250 μg/L LPS的诱导组； 诱导组基础上加100 μg/L雷帕霉素的干预组；干预组基础上加rTNF-α 50 μg/L的抗干预组；及抗干预组基础上加抗鼠TNF-α 100 μg/L的抗体中和组。培养4 h后，ELISA法检测对照组、诱导组和干预组上清液中TNF-α水平；培养24 h后，RT-PCR和Western blotting法分别检测各组细胞内HMGB1 mRNA表达水平和上清液中HMGB1含量。结果:培养4 h，干预组上清液中TNF-α水平与对照组比无显著差异（P>0.05），但明显低于诱导组（P<0.05）；培养24 h, 与对照组比，诱导组细胞内HMGB1 mRNA表达明显增强（P<0.05），上清液中HMGB1含量也明显增多（P<0.05）；干预组明显减少了HMGB1 mRNA表达及上清液中HMGB1含量（P<0.05）； 与干预组比，抗干预组细胞HMGB1 mRNA表达及上清液中HMGB1含量明显增加（P<0.05）；抗体中和组细胞HMGB1 mRNA表达水平及上清液中HMGB1含量和干预组无显著差异（P>0.05）。 结论:雷帕霉素抑制LPS诱导RAW264.7细胞表达和释放HMGB1，可能部分地与其抑制TNF-α的表达有关。     

全蝎水提物对巨噬细胞活化作用研究

目的:观察全蝎水提物(Buthus martensii water extract,BMWE)在体外对小鼠巨噬细胞株RAW264.7的活化作用。方法:MTT法测定细胞活性;采用FITC标记的酵母测定吞噬活性;利用Griess试剂和ELISA方法测定细胞上清NO、TNF-α和IL-6释放量。结果:BMWE在不影响细胞活力的剂量范围内可增强正常及免疫抑制的RAW264.7细胞吞噬荧光酵母能力(P0.01),增加RAW264.7细胞NO释放量(P0.01),促进RAW264.7细胞TNF-α和IL-6产生(P0.01),均具有良好的量效关系。结论:BMWE可明显提高巨噬细胞吞噬功能与分泌能力,活化巨噬细胞,此作用与全蝎传统用于治疗疮疡、瘰疬,现代用于肿瘤的临床应用相关。     

The effects of CpG DNA on HMGB1 release by murine macrophage cell lines

DNA containing cytosine-guanine dinucleotide (CpG) motifs (CpG DNA) has potent immunostimulatory activities that resemble those of lipopolysaccharide (LPS) in its effects on the innate immune system. Among its activities, LPS can induce the release of high mobility group protein (HMGB1) by macrophages, a dual function molecule that can mediate the late effects of LPS. To determine whether CpG DNA can also induce HMGB1 release, the effects of a synthetic CpG oligonucleotide (ODN) on HMGB1 release from RAW 264.7 and J774A.1 cells were assessed by Western blotting of culture supernatants. Under conditions in which the CpG ODN activated the cell lines, as assessed by stimulation of tumor necrosis factor alpha and interleukin-12, it failed to cause HMGB1 release into the media. Although unable to induce HMGB1 release by itself, the CpG ODN nevertheless potentiated the action of LPS. With RAW 264.7 cells, lipoteichoic acid and polyinosinic-polycytidylic acid, like LPS, stimulated HMGB1 release as well as cytokine production. These results indicate that the effects of CpG DNA on macrophages differ from other ligands of Toll-like receptors and may lead to a distinct pattern of immune cell activation in the context of infection or its use as an immunomodulatory agent.     

含CpG基序的寡核苷酸对树突状细胞的作用及其机制

树突状细胞(dendriticcells,DC)是目前所知功能最强的抗原提呈细胞(antigenpresentingcells,APC),其最大特点在于能激活初始型T细胞(naiveTcell),是机体免疫应答的始动者,在免疫应答中起着重要的作用。DC的成熟、活化是其发挥免疫作用的先决条件,因而成为研究的焦点。目前已发现刺激DC成熟的多种因素,包括肿瘤坏死因子-α(tumornecrosis factor-α,TNF-α)、CD40-CD40L、脂多糖(lipopolysaccharide,LPS)和含有非甲基化胞嘧啶鸟嘌呤二核苷酸(cytosinephosphate-guanosine,CpG)基序的寡核苷酸(CpGmotif-containingoligodeoxynucle …     

Rab7负向调控巨噬细胞中CpG诱导的细胞因子的表达

目的:研究Rab7过表达及失活突变(Rab7T22N)对CpG刺激的RAW264.7巨噬细胞中分泌细胞因子的影响。方法:采用RT-PCR和Real-time PCR检测RAW264.7细胞中Rab7在CpG刺激下表达模式。将Rab7真核表达质粒及失活突变质粒Rab7T22N通过脂质体法转染RAW264.7细胞,G418稳定筛选,Western法鉴定表达效果。用CpG刺激稳定表达Rab7的RAW264.7细胞系,RT-PCR和Real-time PCR检测细胞因子IL-6、IL-1β、IFN-β的表达量变化。结果:CpG刺激RAW264.7后,Rab7 mRNA表达水平逐渐增高,在8小时达到高峰,表达增高近4倍。Rab7过表达后,CpG刺激后产生的IL-6、IL-1β、IFN-β显著降低。巨噬细胞中Rab7失活突变后,在CpG刺激后IL-6、IL-1β、IFN-β的表达又显著增加。结论:CpG促进RAW264.7巨噬细胞中Rab7 mRNA的表达,Rab7抑制了CpG刺激的巨噬细胞中IL-6、IL-1β、IFN-β的表达,该抑制作用的发挥与其酶活性的GTP结合有关。该研究为进一步阐明Rab7在CpG/TLR9信号通路中的作用奠定了基础。     

Competition by inhibitory oligonucleotides prevents binding of CpG to C‐terminal TLR9

TLR9 recognizes unmethylated CpG‐containing DNA commonly found in bacteria. Synthetic oligonucleotides containing CpG‐motifs (CpG ODNs) recapitulate the activation of TLR9 by microbial DNA, whereas inversion of the CG dinucleotide within the CpG motif to GC (GpC ODNs) renders such ODNs inactive. This difference cannot be attributed to binding of ODNs to the full‐length TLR9 ectodomain, as both CpG and GpC ODNs bind comparably. Activation of murine TLR9 requires cleavage into an active C‐terminal fragment, which binds CpG robustly. We therefore compared the ability of CpG and GpC ODNs to bind to full‐length and C‐terminal TLR9, and their impact on the cleavage of TLR9. We found that CpG binds better to C‐terminal TLR9 when compared with GpC, despite comparably low binding of both ODNs to full‐length TLR9. Neither CpG nor GpC ODNs affected TLR9 cleavage in murine RAW 264.7 cells stably expressing TLR9‐Myc. Inhibitory ODNs (IN‐ODNs) block TLR9 signaling, but how they do so remains unclear. We show here that inhibitory ODNs do not impede TLR9 cleavage but bind to C‐terminal TLR9 preferentially, and thereby compete for CpG ODN binding both in RAW cells and in TLR9‐deficient cells transduced with TLR9‐Myc. Ligand binding to C‐terminal fragment thus determines the outcome of activation through TLR9.     

The Effect of Epigallocatechin Gallate on Lipopolysaccharide-Induced Acute Lung Injury in a Murine Model

This study was performed to evaluate the effects of epigallocatechin 3 gallate (EGCG) on lipopolysaccharide (LPS)-induced acute lung injury in a murine model. In the present study, production of TNF-α and MIP-2 and activation of extracellular signal-regulated kinases (ERK)1/2, c-Jun amino terminal kinases (JNK) and p38 in RAW264.7 cells were measured. EGCG inhibited the production of TNF-α and MIP-2, and attenuated phosphorylation levels of ERK1/2 and JNK, but not p38 in RAW264.7 cells stimulated with LPS. Also, EGCG attenuated the production of TNF-α and MIP-2, and the phosphorylation of ERK1/2 and JNK in the lungs of mice administered with LPS intratracheally. It reduced wet/dry weight ratio, histological severities, and neutrophil accumulation in the lungs in mice given LPS. Our results showed that EGCG attenuated LPS-induced lung injury by suppression of the MIP-2 and TNF-α production, and ERK1/2 and JNK activation in macrophage stimulated with LPS.     

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.     

Specific siRNA downregulated TLR9 and altered cytokine expression pattern in macrophage after CpG DNA stimulation

Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)candirectly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptorTLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial tothe subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are notfully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokineprofiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid uponCpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased whileTLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6,IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alterationof cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role ofCpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology.2005;2(2):130-135.     

Role of the heat shock protein 90 in immune response stimulation by bacterial DNA and synthetic oligonucleotides

To elucidate the mechanisms of immunostimulation by bacterial DNA and synthetic oligonucleotides, the effects of heat shock protein 90 (Hsp90) inhibitors on the activation of murine spleen cells and macrophages by these molecules were investigated. Murine spleen cells and J774 and RAW264.7 macrophages responded to a CpG-containing oligodeoxynucleotide (CpG ODN) and Escherichia coli DNA by increased production of interleukin 6 (IL-6), IL-12, tumor necrosis factor alpha, and nitric oxide (NO). Pretreatment with any of the three Hsp90 inhibitors geldanamycin, radicicol, and herbimycin A resulted in a dose-dependent suppression of cytokine production from the spleen cells and macrophages and of NO from macrophages stimulated with CpG ODN or E. coli DNA. These Hsp90 inhibitors, however, had no effect on Staphylococcus aureus Cowan strain 1-induced IL-12 production from either the murine spleen cells or macrophages. CpG ODN and E. coli DNA induced increased intracellular levels of phosphorylated extracellular signal-regulated kinases (ERK1 and -2), which are members of the mitogen-activated protein (MAP) kinase family, while geldanamycin and radicicol blocked the phosphorylation of ERK1 and -2 in J774 and RAW264.7 cells. These data indicate that DNA-induced activation of murine spleen cells and macrophages is mediated by Hsp90 and that Hsp90 inhibitor suppression of DNA-induced macrophage activation is associated with disruption of the MAP kinase signaling pathway. Our findings suggest that Hsp90 inhibitors may provide a useful means of elucidating the mechanisms of immunostimulation by bacterial DNA and CpG ODN as well as a strategy for preventing adverse effects of bacterial DNA as well as lipopolysaccharide.     

葫芦素E对脂多糖诱导RAW264.7巨噬细胞炎症反应的影响及作用机制

分析葫芦素E(CuE)对脂多糖(LPS)诱导的小鼠RAW264.7巨噬细胞炎症反应的影响,研究其抗炎作用的分子机制。采用改良MTT法检测RAW264.7细胞的增殖;以碘化丙锭染色检测CuE对细胞周期的影响;采用细胞内细胞因子染色法分析肿瘤坏死因子(TNF-α)的表达;免疫荧光染色分析胞内Actin的结构;应用免疫印迹法检测CuE对G-肌动蛋白(G-ac-tin)及核转录因子NF-κB核转位的影响。实验结果显示CuE对RAW264.7细胞的增殖具有剂量依赖性抑制作用,并降低细胞内G-actin的水平;CuE明显阻止LPS诱导的细胞伸展和伪足形成,使细胞周期阻滞于G2/M期。同时,CuE还明显抑制LPS活化的RAW264.7细胞表达促炎因子TNF-α,并显著降低LPS诱导的转录因子NF-κB的核转位作用。这些结果表明,CuE通过破坏RAW264.7巨噬细胞的肌动蛋白细胞骨架,引起细胞周期阻滞,并抑制LPS诱导的NF-κB核转位以及TNF-α的表达,从而发挥抗炎作用。