Role of protein kinase C in hydroxyapatite- induced phagocytosis by a murine macrophage cell line (RAW264.7)

The role of protein kinase C (PKC) in hydroxyapatite (HA)-induced phagocytosis by RAW 264.7 cells was investigated. The cells were incubated with HA particles at various incubation time and the levels of PKC activity were determined from the cell lysate. To determine the role of PKC, particles were incubated with the cells pretreated with the various concentrations of bisindolylmaleimide, a PKC inhibitor, and phagocytosis was then assessed at 60 min. Latex beads were used as a control. Our results showed that following incubation with HA particles, the levels of PKC activity in RAW264.7 cells was highest at 7 min and then decreased to reach the baseline levels of the controls at 30 min. Pretreatment of the cells with bisindolylmaleimide significantly reduced phagocytosis of HA particles in a dose-dependent pattern. The results of our present study suggest therefore that ingestion of HA by RAW264.7 cells may depend on PKC activity that may act in the early stages of phagocytosis.     

Biological effect of gamma-rays and LPS on the murine macrophage cell line RAW264.7 in mice

AIM: To explore the effect of activating the murine macrophage cell line RAW264.7 by both gamma-rays and lipopolysaccharide (LPS) and to study the expression of calcium-binding protein S100A8 induced by gamma-rays and LPS. METHODS: The RAW264.7 cells were observed by phase contrast microscope. The cell cycle and the level of reactive oxygen intermediates (ROIs) were detected by flow cytometry (FCM). The production of NO was measured by colorimetric Griess reaction. The mRNA expression of S100A8 was recorded by real-time quantitative RT-PCR method. RESULTS: The exposure of RAW264.7 cells to gamma-rays and LPS resulted in the morphological change of cells, the rise of cells number of aneuploid and apoptosis, and the rise of the level of ROI, NO and S100A8 mRNA. The effect of using both gamma-rays and LPS was stronger than that of single gamma-rays or LPS treatment. CONCLUSION: The mechanism of using both gamma-rays and LPS for activating macrophages is owing to the various biological effects including the change of cell cycle, the change of the level of messenger molecules and the expression of inflammation factor such as S100A8. The expression of S100A8 gene is closely correlated with the function and state of macrophages.     

γ射线与LPS作用于小鼠巨噬细胞RAW264.7的生物学效应

目的: 研究γ射线与LPS协同激活小鼠巨噬细胞RAW264.7的效应机制, 以及γ射线与LPS诱导钙结合蛋白S100A8的表达及意义.方法: 相差显微镜观察细胞形态; 流式细胞计数方法检测细胞周期及活性氧介质(ROI)水平; Griess颜色反应测定细胞NO水平; 实时定量RT-PCR方法检测细胞S100A8 mRNA水平的表达.结果: γ射线与LPS作用于RAW264.7细胞引起细胞形态改变, 部分细胞出现非整倍性, 少量细胞出现凋亡, 胞内ROI水平、 NO水平明显升高, S100A8 分子mRNA水平明显升高, 而且这些效应几乎都比?射线或LPS单因素的作用要强.结论: γ射线与LPS协同诱导巨噬细胞激活是细胞周期改变、信使分子水平变化、炎症因子如S100A8的表达等多方面生物效应的综合结果, 其中S100A8基因的表达与巨噬细胞功能状态密切相关.     

Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.     

Lipopolysaccharide induces calcitonin gene-related peptide in the RAW264.7 macrophage cell line

Calcitonin gene‐related peptide (CGRP) is widely distributed and plays important roles in a wide array of biological functions. It is enriched in primary sensory neurons and hence involved in nociception and neurogenic inflammation. Recent studies have shown that CGRP can be produced by immune cells such as monocytes/macrophages following inflammatory stimulation, suggesting a role in innate immunity. However, it is unclear how CGRP is up‐regulated in macrophages and if it plays a role in macrophage functions such as the production of cytokines and chemokines. Using enzyme‐linked immunosorbent assay (ELISA) and multiplex ELISA, lipopolysaccharide (LPS) was found to induce CGRP in the RAW 264.7 macrophage cell line. LPS‐induced inflammatory mediators such as nerve growth factor (NGF), interleukin‐1β (IL‐1β), IL‐6, prostaglandin E₂ (PGE₂) and nuclear factor‐κB (NF‐κB) signalling are involved in inducing CGRP, whereas the NGF receptor trkA and CGRP receptor signalling pathways are unexpectedly involved in suppressing LPS‐induced CGRP, which leads to the fine‐tune regulation of CGRP release. Exogenous CGRP and CGRP receptor antagonists, in a concentration‐dependent manner, stimulated, inhibited or had no effect on basal or LPS‐induced release of monocyte chemoattractant protein‐1, IL‐1β, IL‐6, tumour necrosis factor‐α and IL‐10 in RAW macrophages. The ligand‐concentration‐dependent regulation of the production of inflammatory mediators by CGRP receptor signalling is a novel mechanism underlying the stimulating and suppressing role of CGRP in immune and inflammatory responses. Together, our data suggest that monocytes/macrophages are an important source of CGRP. Inflammation‐induced CGRP has a positive or negative reciprocal effect on the production of other pro‐ and anti‐inflammatory mediators. Thereby CGRP plays both facilitating and suppressing roles in immune and inflammatory responses.     

Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响

探讨viili胞外多糖(Viili exopolysaccharides,VEPS)对小鼠巨噬细胞RAW264.7激活和增殖的影响。噻唑蓝(MTT)比色法检测细胞的生长与增殖;中性红吞噬实验检测吞噬活性;Griess试剂盒检测培养上清液中NO分泌量,ELISA法检测VEPS不同浓度及不同作用时间培养上清中IL-6,IL-1β含量;扫描电子显微镜观察VEPS对细胞形态的影响;碘化丙啶(PI)染色检测VEPS对细胞周期的影响。结果显示,VEPS对RAW264.7细胞的增殖、吞噬能力、分泌NO、IL-6、IL-1β等都有显著的促进作用,VEPS为100μg/ml时促进作用最明显,呈剂量相关,作用72h时细胞因子分泌量达到最大,72h后下降。VEPS激活巨噬细胞并使其变得扁平伸展且形成伪足。VEPS促进G1期细胞增多,提高细胞的增殖能力。VEPS免疫调节作用与其激活RAW264.7细胞,促进NO、IL-6、IL-1β等分泌有关,且VEPS与LPS对RAW264.7细胞有相似的作用规律。以上结果证明VEPS能激活巨噬细胞,也可能最终激活淋巴细胞,达到增强非特异性和特异性免疫的作用。     

干扰盐皮质激素受体基因对RAW 264.7细胞增殖和凋亡的影响

 目的：应用RNA干扰技术沉默小鼠RAW 264.7巨噬细胞盐皮质激素受体(MR)基因,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法：针对MR基因设计合成重组MR shRNA质粒,脂质体法转染质粒至RAW 264.7细胞,经G418筛选后获得稳定表达细胞株。细胞分为3组：野生型(WT)组、阴性对照（NC）组和干扰（shMR）组。荧光显微镜下观察确定细胞的转染效率;实时定量PCR法检测细胞中MR mRNA的表达;CCK-8方法检测细胞增殖活性;流式细胞术分析细胞周期分布和凋亡情况。结果：(1) MR shRNA能明显抑制RAW 264.7细胞的MR基因表达,抑制率70%以上。(2) 从第3 d开始,shMR组细胞的生长速度明显低于NC和WT组（P<0.05）,说明MR shRNA能明显抑制细胞增殖。(3) WT、NC和shMR组的增殖指数分别为(37.2±0.5)%、(37.5±1.6)%和(31.0±1.3)%,shMR组的细胞周期出现改变,S期和G₂/M期比例明显下降,增殖指数下降（P<0.05）。(4) WT、NC和shMR组的细胞凋亡率分别为(2.18±0.36)%、(6.65±0.81)%和(7.70±1.34)%,shMR组略高于NC组,但二者的差异无统计学意义（P>0.05）。结论：本研究成功构建了稳定干扰MR基因表达的RAW 264.7细胞株,MR shRNA能够明显抑制RAW 264.7细胞增殖,但对其凋亡无明显影响。     

激活素A对RAW264.7巨噬细胞活性的调节作用

目的探讨激活素A对参与炎症反应的小鼠巨噬细胞活性调节作用。方法以LPS刺激活化的小鼠巨噬细胞系RAW264．7细胞作为阳性参照,ELISA法检测激活素A及LPS刺激的小鼠腹腔巨噬细胞系RAW264．7细胞IL-1β分泌水平,还原酶法分析NO分泌水平,RT-PCR检测IL-1β和iNOS mRNA的表达,瑞氏染色检测RAW264．7细胞吞噬活性。结果在激活素A刺激下RAW264．7细胞IL-1β和NO分泌水平均明显升高,IL-1β和iNOS mRNA表达亦增加,巨噬细胞吞噬活性增强;激活素A和LPS共刺激RAW264．7细胞时,激活素A明显抑制LPS刺激的RAW264．7细胞IL-1β和NO产生水平,以及IL-1β和iNOS mRNA表达,巨噬细胞吞噬活性也明显低于LPS单独刺激组。结论激活素对巨噬细胞的活性调节具有双重作用,这种作用与巨噬细胞的激活状态有关。     

鼠巨细胞病毒感染RAW264.7细胞的体外实验研究

目的 体外观察鼠巨细胞病毒(murine cytomegalovirus,MCMV)对鼠巨噬细胞株RAW264.7的感染特点及其对细胞增殖和凋亡的影响.方法 分别以感染复数(multiplicity of infection,MOI)为1、0.1、0.01的MCMV Smith株感染RAW细胞,于感染后6、12、24、36、48、72、96和120 h收集细胞和培养上清,光镜下观察细胞病变,透射电镜观察感染细胞内病毒颗粒和细胞超微结构变化;免疫细胞化学检测MCMV早期抗原(early antigen,EA)表达以及蚀斑形成实验监测病毒增殖情况;MTT法和流式细胞术分别评估感染细胞增殖与凋亡的改变,以MCMV敏感的鼠胚胎成纤维细胞为阳性对照.结果 MCMV感染后24～48 h可见RAW细胞肿胀和脱落,电镜显示RAW胞浆中存在完整的病毒颗粒且细胞器肿胀明显;病毒感染RAW细胞后6 h(MOI:1和0.1)～12 h(MOI=0.01)可见病毒EA的阳性表达;感染后24 h培养上清中病毒滴度即明显上升,至96～120 h达高峰;RAW细胞增殖在感染后72～120 h受到明显抑制;当MOI=0.1时,感染后72～120 h细胞死亡率明显增高,但凋亡率改变不显著.结论 巨噬细胞株RAW264.7在体外对MCMV易感,较鼠胚胎成纤维细胞更快形成溶细胞性的产毒性感染;病毒可抑制细胞增殖但不影响细胞凋亡,为深入探讨CMV相关免疫学致病机制提供了一个良好的体外细胞模型.     

青藤碱对LPS、IL-4诱导的小鼠RAW264.7巨噬细胞极化的影响

目的:探讨青藤碱(Sinomenine,SIN)对脂多糖(LPS)以及白细胞介素4(IL-4)诱导的RAW264.7细胞向M1、M2型极化的影响。方法:以LPS刺激RAW264.7细胞诱导M1型极化,IL-4刺激RAW264.7细胞诱导M2型极化;青藤碱作用于LPS或IL-4诱导的巨噬细胞后:用酶联免疫法(ELISA)检测不同诱导状态下RAW264.7细胞TNF-α和IL-10的分泌量;荧光定量PCR检测与巨噬细胞极化相关的精氨酸酶-1(Arg-1)、一氧化氮合酶(i NOS)、细胞因子信号转导抑制蛋白-2(SOCS2)和细胞因子信号转导抑制蛋白-3(SOCS3)的mRNA表达水平。结果:青藤碱能抑制LPS诱导下细胞TNF-α的分泌量,抑制细胞i NOS和SOCS3的mRNA表达水平的升高。青藤碱能抑制IL-4诱导下细胞IL-10的分泌量和Arg1的mRNA表达水平的升高,对IL-4诱导下细胞SOCS2的mRNA表达水平的升高没有明显影响。结论:青藤碱对LPS诱导下巨噬细胞向M1型极化具有抑制作用;对IL-4诱导下巨噬细胞向M2型极化具有抑制作用。青藤碱对M1/M2亚型的失衡具有调节作用,有利于维持其动态平衡。     

Transforming growth factor-beta 1 inhibits activation of macrophage cell line RAW 264.7 for cell killing.

Transforming growth factor beta-1 (TGF-beta) is a multi-potent immunoregulatory peptide that has effects on numerous cell types. Here we report that human TGF-beta inhibits the activation of the macrophage cell line RAW 264.7 for killing of the L1210 tumour cell line. RAW 264.7 cells, like normal macrophages, require sequential interaction with priming and triggering stimuli for full activation of cytolytic activity. TGF-beta inhibits this cytotoxicity in a dose-dependent manner at both the priming and the triggering stage. Addition of as little as 1 ng/ml TGF-beta when added with either the priming signal, recombinant interferon-gamma (IFN-gamma), or the triggering signal, bacterial lipopolysaccharide (LPS), completely abrogated tumouricidal activity. Incubation with TGF-beta also inhibited the morphological changes normally observed in activated RAW 264.7 cells. However, TGF-beta was unable to inhibit the cytotoxic activity of RAW 264.7 cells against the target cell line WEHI 164, which is sensitive to tumour necrosis factor. In contrast to the effects on cytotoxic activity, the cytostatic activity of activated RAW 264.7 cells was not inhibited by TGF-beta at doses of up to 5 ng/ml. In addition, pretreatment of the L1210 target cells with TGF-beta made them refractory to both the cytostatic and cytotoxic effects of RAW 264.7 cells. These data suggest that TGF-beta may be an important mediator in the regulation of macrophage tumouricidal activity.     

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.     

Expression of murine N-MYC by insertion of retrovirus sequences in a murine macrophage cell line (RAW264).

RAW264 cells, reported to be originated from Abelson-virus-induced B lymphomas, are widely used as a murine monocyte cell line. We found that RAW264 show enhanced expression of murine N-MYC. Murine cDNA clones associated with N-MYC were separated from (lambda)gt11 cDNA library constructed by using mRNA from the macrophage cell line, RAW264 cells. Sequencing analysis of the longest cDNA clone N-MYCL showed that the length of the coding region was 18 bases shorter than that of the predicted full length N-MYC cDNA, and the 3' untranslated region had the 5' long terminal repeat (LTR) sequence of the Moloney-like proviral sequence, suggesting the expression of N-MYC by insertion of the proviral sequence. This suggests that expression of N-MYC plays a role in the establishment of macrophage cell line RAW264. Integration of LTR and overexpression of the N-MYC gene might have existed in the parental lymphoma cells, playing a role in the development of lymphoma or in the establishment of macrophage cell line.     

干扰P2X_7R基因对RAW264.7细胞增殖和吞噬的影响

目的:应用RNA干扰技术抑制小鼠巨噬细胞RAW264.7细胞P2X7受体(P2X7R)基因的表达,建立稳定干扰细胞株,并观察其对细胞增殖和凋亡的影响。方法:用脂质体法将P2X7R shRNA重组质粒转染至RAW264.7细胞,经G418筛选后获得稳定干扰细胞株。细胞分为野生型(WT)组、阴性对照(NC)组和干扰(sh P2X7R)组。Real-time PCR法检测细胞中P2X7R mRNA的表达,Western blot检测细胞中P2X7R蛋白的表达;CCK-8方法检测细胞生长活性,5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine,Ed U)掺入实验检测细胞增殖活性;流式细胞术分析细胞周期的分布和吞噬情况。结果:P2X7R shRNA能明显抑制RAW264.7细胞的P2X7R mRNA和蛋白的表达,抑制率在80%以上。48 h后,sh P2X7R组细胞的生长速度明显高于NC组和WT组(P0.05),增殖期细胞比例明显升高(P0.05),说明下调P2X7R基因能明显促进细胞增殖。sh P2X7R组的细胞周期出现改变,S期和G2/M期的比例明显上升,增殖指数增高(P0.05)。sh P2X7R组的细胞吞噬活性明显高于NC组(P0.05)。结论:本研究成功构建了稳定干扰P2X7R基因表达的小鼠巨噬细胞株RAW264.7,sh P2X7R能够明显促进RAW264.7细胞的增殖,改变了细胞的吞噬活性。     

The effect of L-arginine on Porphyromonas gingivalis-induced phagocytosis of a murine macrophage-like RAW264.7 cell line

The aim of this study was to determine the effect of L-arginine on Porphyromonas gingivalis-induced phagocytosis by RAW264.7 cells. The cells were pretreated with L-arginine or D-arginine prior to incubation with either unopsonized or opsonized P. gingivalis. In other experiments, the cells were pretreated with L-arginine and various concentrations of NMLA (N(G)-monomethyl-L-arginine) prior to incubation with the bacteria. The phagocytosis was microscopically assessed and determined by the phagocytic index. The results showed that L-arginine, but not D-arginine enhances the ability of RAW264.7 cells to engulf the bacteria. The upregulatory effect of L-arginine on P. gingivalis-induced phagocytosis was abolished by NMLA. The results of the present study suggest that L-arginine may upregulate the P. gingivalis-induced phagocytic activity of RAW264.7 cells, perhaps, via modulation of nitric oxide synthase.     

双氢青蒿素抑制核因子κB受体激动剂配体诱导RAW264.7细胞分化破骨细胞的研究

目的 探讨双氢青蒿素(DA)对核因子κB受体激动剂配体(RANKL)诱导RAW264.7细胞形成破骨细胞的影响。方法 通过细胞计数试剂盒(CCK-8)确定不同浓度(1、5、10、50、100、200 μmol/L)DA对RAW264.7细胞的生存影响。分别用50 ng/mL RANKL及50 ng/mL RANKL+5、10、50、100 μmol/L DA诱导RAW264.7细胞形成破骨细胞,共3 d。对形成的破骨细胞用抗酒石酸酸性磷酸酶(TRAP)染色并计数,TRAP染色阳性且细胞核数目＞3个认为是成熟的破骨细胞。50 ng/mL RANKL及50 ng/mL RANKL+10、100 μmol/L DA培养RAW264.7细胞24 h,用Trizol试剂提取总RNA,并使用荧光实时定量PCR检测破骨细胞分化相关基因NFATc1、c-fos及Cathepsin K的表达。结果 1、5、10、50、100 μmol/L DA对RAW264.7细胞毒性较小,细胞存活率均＞90%。TRAP染色显示,5、10、50、100 μmol/L DA可以减少RANKL诱导成熟破骨细胞的数目,并呈剂量依赖关系(F=139.156, P＜0.01)。实时荧光定量PCR结果显示,10、100 μmol/L DA具有下调破骨细胞分化关键基因NFATc1和c-fos表达的作用,且100 μmol/L抑制作用比10 μmol/L明显,但两种浓度DA对Cathepsin K的表达无明显影响。结论 DA对RAW 264.7细胞毒性较低,通过下调RAW 264.7细胞NFATc1和c-fos基因表达抑制RANKL诱导破骨细胞形成。DA可以作为治疗骨质破坏性疾病的潜在药物。