核转录因子Sp1在二氧化硅活化的Ⅱ型肺泡细胞中的表达及作用

目的研究二氧化硅(SiO2)刺激的Ⅱ型肺泡细胞(A549)中核转录因子Sp1表达和定位的动态变化,探讨其在矽肺发生发展中的作用。方法复制SD大鼠矽肺模型,免疫组化检测体内SiO2刺激的Ⅱ型肺泡细胞中核转录因子Sp1蛋白表达的定位及其动态变化;逆转录-聚合酶链反应检测体外SiO2刺激的A549细胞中核转录因子Sp1mRNA的动态变化;免疫细胞化学、Western印迹检测体外SiO2刺激的A549细胞中核转录因子Sp1蛋白表达的定位及其动态变化。结果大鼠矽肺模型中,SiO2刺激组与空白对照组相比,Ⅱ型肺泡细胞中核转录因子Sp1蛋白表达上调,于第7~14天达高峰;体外SiO2作用A549细胞30~960min,Sp1mRNA表达呈现先升后降,峰值位于60min;Sp1总蛋白和核蛋白表达亦呈现先升后降的趋势,总蛋白表达峰值位于120min,核蛋白峰值位于240min;Sp1蛋白于60min开始发生明显的核移位,240min时核移位最明显。结论SiO能通过增强Ⅱ型肺泡细胞(A549)中Sp1的表达和核移位,从而在矽肺的发生发展过程中起重要的作用。     

二氧化硅活化巨噬细胞中早期生长反应因子-1及其信号转导通路的研究

目的 探讨早期生长反应因子(Egr-1)及其信号转导在矽肺发生发展中的作用。方法用细胞免疫荧光、原位杂交方法检测二氧化硅(SiO2)刺激后Egr-1的表达定位,用报道质粒及EMSA检测其活性改变;用激酶活性分析法检测si0：刺激巨噬细胞后ERK1／2活性改变,进一步用激酶抑制剂初步探讨SiO2活化Egr-1的信号转导通路。结果SiO2刺激RAW264．7细胞短时间Egr-1核蛋白表达及转录因子明显增加;且在处理后30～60min,Egr-1核蛋白结合活性明显升高(为未处理组的20倍);在刺激后15min ERK1／2活性开始升高,30min达高峰(活性为对照组的29倍)而后渐降至基础水平;进一步用激酶阻断发现,Egr-1 mRNA及蛋白表达均减少。结论SiO2能激活巨噬细胞中Egr-1,且此过程可能由ERK1／2、p38介导,提示SiO2-ERK1／2、p38-Egr-1通路可能在矽肺发生发展过程中起重要作用。     

核转录因子在二氧化硅刺激巨噬细胞产生细胞因子中的作用

目的 探讨二氧化硅(SiO2)刺激巨噬细胞生成肿瘤坏死因子(TNF)-α、转化生长因子(TGF)-β1增多的分子机制及核转录因子Egr-1和NF-κB介导的信号通路在硅肺发生发展中的作用。方法 Egr-1或NF-κB抗体和反义寡核苷酸分别处理巨噬细胞后,用ELISA法检测细胞上清液中TNF-α蛋白的含量;免疫细胞化学链霉素抗生物素蛋白-过氧化物酶(SP)法检测细胞中TGF-β1蛋白的表达;逆转录-聚合酶链反应(RT-PCR)检测TNF-α和TGF-β1mRNA的表达。结果 与非抗体处理组比较,SiO2刺激下抗体处理组巨噬细胞生成TNF-α蛋白和TGF-β1蛋白减少,其mRNA表达也相应下降(P<0.05);与未转染和转染正义寡核苷酸的细胞相比,Egr-1或NF-κB反义寡核苷酸转染细胞后,在SiO2刺激下巨噬细胞生成TNF-α蛋白和TGF-β1蛋白减少,二者mRNA表达也相应下降(P<0.05)。结论 SiO2刺激巨噬细胞生成TNF-α和TGF-β1可能主要是经核转录因子Egr-1和NF-κB介导,Egr-1与NF-κB抗体和反义寡核苷酸的应用可能会导致早期肺泡炎的活动性下降,延缓或阻止肺纤维化的形成。     

内毒素体外诱导巨噬细胞炎症蛋白-2γ的表达

目的：体外研究内毒素对巨噬细胞炎症蛋白—2γ(MIP-2γ)表达的作用。方法：选用小鼠单核-巨噬细胞株RAW264．7及原代培养的BALB／c小鼠肾脏细胞用LPS刺激，以实时荧光定量RT—PCR检测不同时间点MIP-2γ mRNA表达水平的变化。结果：RAW264．7细胞在LPS刺激后2h，MIP-2γ mRNA表达水平快速到达峰值(较正常水平增加约7倍)，以后缓慢下降，至刺激后16h基本恢复正常水平。原代培养肾脏细胞经LPS刺激后12h，MIP-2γ mRNA表达水平增长约50倍。结论：LPS在体外可明显诱导单核-巨噬细胞和肾脏细胞MIP-2γ mRNA的表达，提示MIP-21参与了炎症过程。     

微管相关蛋白1轻链3及Beclin-1在硅沉着病大鼠肺泡巨噬细胞中的表达

目的观察自噬相关蛋白——微管相关蛋白1轻链3(LC-3)、Beclin-1在硅沉着病大鼠肺泡巨噬细胞中的表达,从细胞自噬角度探讨硅沉着病形成的分子机制。方法 50只成年雄性SD大鼠随机分为2组(n=25):对照组和硅沉着病模型组,每组再分5个时相组。采用非暴露气管灌注二氧化硅(SiO2)粉尘混悬液法(50g/L)建立大鼠硅沉着病模型。分别于造模后1、3、7、14及28 d分批处死5只大鼠,进行原位支气管肺泡灌洗,获取肺泡巨噬细胞,进行培养纯化和富集后用于后续研究。HE染色及透射电子显微镜观察肺泡巨噬细胞形态学改变;免疫细胞化学法检测LC-3、Beclin-1的表达及分布;免疫印迹法检测LC-3、Beclin-1的蛋白表达量。结果与对照组相比模型组肺泡巨噬细胞体积较大,胞质丰富,部分细胞内可见硅沉着吞噬颗粒,电镜下可见自噬体形成;模型组LC-3、Beclin-1在各时间点的表达较对照组均增多(P0.05),1 d即开始增多,随着时间的延长表达逐渐增多,至14 d时达高峰(P0.05),28d时回落,但仍高于对照组的表达。结论在硅沉着病大鼠肺泡巨噬细胞中有自噬的激活,肺泡巨噬细胞自噬参与了大鼠硅沉着病的病理进程。     

嗜肺军团菌MOMP通过激活NOD2/RIP2信号通路抑制RAW264.7巨噬细胞的吞噬功能并增强其趋化能力

目的探讨嗜肺军团菌主要外膜蛋白(MOMP)对RAW264. 7巨噬细胞吞噬功能和趋化功能的影响并探讨其机制。方法采用MOMP与RAW264. 7巨噬细胞进行体外共培养,用CCK-8法检测MOMP对RAW264. 7巨噬细胞的毒性,确定半数抑制浓度(IC50)。采用(1. 14、0. 57、0. 28)μg/m L MOMP分别处理RAW264. 7巨噬细胞,并设细胞对照组。RAW264. 7巨噬细胞处理24、48、72 h,收集细胞和培养上清,用中性红吞噬实验检测巨噬细胞的吞噬功能;用TranswellTM小室检测巨噬细胞的趋化功能; ELISA检测细胞培养上清单核细胞趋化蛋白1(MCP-1)和白细胞介素10(IL-10)的含量;实时定量PCR检测巨噬细胞核苷酸结合寡聚结构域1(NOD1)、NOD2、受体相互作用蛋白2(RIP2) mRNA水平,Western blot法检测NOD1、NOD2、RIP2的蛋白水平。结果 CCK-8法检测MOMP对RAW264. 7巨噬细胞的IC_(50)为5. 69μg/m L;与对照细胞相比,MOMP处理引起RAW264. 7巨噬细胞吞噬功能降低且呈剂量和时间依赖性;随着MOMP剂量的增加,巨噬细胞的趋化能力及细胞培养上清中MCP-1、IL-10的分泌水平增加,并在36 h达到峰值; NOD2、RIP2的mRNA和蛋白表达水平也增加,NOD2和RIP2的mRNA水平在12 h达到高峰,蛋白水平在24 h达到峰值。结论 MOMP抑制RAW264. 7巨噬细胞的吞噬功能并增强其趋化功能,与激活NOD2/RIP2信号通路有关。     

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。     

IL-17A通过上调ABCA1的表达减少RAW264.7巨噬细胞脂质的积聚

目的:探讨白细胞介素17A(IL-17A)对RAW264. 7巨噬细胞三磷酸腺苷结合盒转运体A1(ABCA1)表达的影响。方法:用不同浓度及的IL-17A处理小鼠RAW264. 7细胞6 h或24 h,或用同一浓度IL-17A处理RAW264. 7细胞不同时间;采用RT-qPCR及Western blot法检测细胞ABCA1的mRNA和蛋白表达情况;采用NBD-胆固醇法检测细胞胆固醇流出情况;采用油红O染色法测定细胞脂质蓄积情况。结果:与对照组相比,IL-17A可以增加RAW264. 7巨噬细胞ABCA1蛋白的表达水平,但不影响ABCA1 mRNA的表达。经IL-17A干预,RAW264. 7巨噬细胞内胆固醇向Apo A-1流出增多,同时细胞脂质蓄积显著减少(P 0. 01)。结论:IL-17A可以增加RAW264. 7巨噬细胞ABCA1的表达水平,其机制并不是通过转录水平实现的,这种影响可能与其抗动脉粥样硬化作用有关。     

黄连解毒汤含药血清对巨噬细胞自噬相关基因表达的影响

目的:考察黄连解毒汤含药血清对RAW264郾7 巨噬细胞自噬的影响,初步探讨其抗动脉粥样硬化的作用机制。方法:采用大、中、小剂量黄连解毒汤水煎剂灌胃干预SD 大鼠,制备含药血清;经MTT 法观察含药血清对细胞增殖的影响后,选择各剂量浓度为20% 的含药血清干预体外培养的巨噬细胞,采用荧光定量PCR 方法检测自噬相关基因Beclin1 和mTOR 的mRNA 表达水平;Western blot 检测Beclin1、p鄄mTOR 蛋白的表达变化。结果:与正常对照组血清相比,黄连解毒汤含药血清干预后诱导了Beclin1 mRNA 及蛋白的表达,抑制了mTOR mRNA 及p鄄mTOR 蛋白的表达。结论:黄连解毒汤含药血清可诱导RAW264郾7 巨噬细胞自噬相关基因Beclin1 的表达、抑制mTOR 的表达,这可能是该方抗动脉粥样硬化作用的重要机制之一。     

siRNA沉默MIF基因对糖皮质激素抑制脂质炎症介质释放的影响及其机制

 目的： 研究小干扰RNA（siRNA）阻断巨噬细胞移动抑制因子（macrophage migration-inhibitory factor,MIF）基因表达对糖皮质激素抑制脂质炎症介质释放的影响及其细胞内机制。方法：体外培养小鼠巨噬细胞系RAW2647,采用免疫荧光法观测siRNA转染效率,RT-PCR检测MIF mRNA的表达,Western blotting检测MIF蛋白的表达;RAW2647细胞转染MIF siRNA后观察地塞米松（Dex）抗炎作用的变化,用ELISA检测细胞上清中前列腺素E2(PGE2)和白三烯B4(LTB4)的含量,Western blotting检测胞浆膜联蛋白Annexin 1和下游胞浆磷酸酯酶A2α（cPLA2α）的蛋白表达变化。结果：与阴性对照相比,MIF siRNA能有效阻断细胞内源性MIF蛋白的表达,增强RAW2647细胞对Dex作用的敏感性;明显增强Dex抑制PGE2和LTB4产生的效应,增加胞浆蛋白Annexin 1的表达,抑制cPLA2α的磷酸化。结论：MIF siRNA能增强糖皮质激素抑制脂质炎症介质PGE2和LTB4的释放,且可能是通过影响Annexin 1-cPLA2α信号通路实现的。阻断内源性MIF蛋白的表达可显著增强RAW2647细胞对糖皮质激素抗炎作用的敏感性。     

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.     

羧胺三唑抑制肿瘤细胞诱导的巨噬细胞中肿瘤坏死因子-α的释放

目的观察体外培养的肿瘤细胞对巨噬细胞炎症因子——肿瘤坏死因子-α(TNF-α)的诱导作用;初步探索羧胺三唑(CAI)对肿瘤诱导的巨噬细胞中TNF-α释放的影响。方法利用transwell装置,建立Lewis肺癌细胞(LLC)与RAW264.7巨噬细胞共培养体系,采用荧光定量PCR方法和ELISA方法分别分析RAW264.7中TNF-α的表达和释放;用LLC条件培养基(LCM)诱导RAW264.7,同时给予CAI处理,采用CCK-8方法分析LCM和CAI对RAW264.7活力的影响,采用ELISA方法分析CAI对诱导后的RAW264.7中TNF-α释放的影响。结果与LLC共培养24 h可显著增加RAW264.7中TNF-α相对表达量[单独培养vs共培养,(1.00±0.12)vs(2.23±0.17),P<0.01]和释放[单独培养vs共培养,(65.21±12.76)vs(143.92±19.22)pg/ml,P<0.05];LCM和CAI在1 h和4 h对RAW264.7活力没有影响,CAI可以显著抑制LCM对RAW264.7中TNF-α释放的诱导(4 h,P<0.01)。结论 LLC肿瘤微环境可以诱导RAW264.7中TNF-α的表达增加;CAI对这种诱导的抑制使其在肿瘤环境中表现出一定的抗炎作用,可能是其抗肿瘤作用的机制之一。     

TIM-1 regulates macrophage cytokine production and B7 family member expression

T-cell immunoglobulin mucin-1 (TIM-1) is associated with the regulation of T helper type 2 (Th2) immune responses and has been associated with asthma susceptibility. Previous studies have shown that administration of TIM-1 results in T cell hyperproliferation and increased Th2 cytokine secretion. TIM-1 has also been shown to bind to macrophages, but the effects of TIM-1 administration on macrophage activity have not been assessed. In this study we demonstrate that TIM-1 binds to the mouse macrophage cell line RAW 264.7. Stimulation of the RAW264.7 cells with TIM-1 increases nitric oxide production. A dramatic increase in the pro-inflammatory cytokines TNF-alpha and IL-6 is seen upon TIM-1 stimulation of RAW 264.7 cells. Additionally, there is a moderate increase in the immuno-modulatory cytokine IL-10 when RAW 264.7 cells are stimulated with TIM-1. TIM-1 stimulation also alters the expression of some members of the B7 family of co-stimulatory/co-inhibitory proteins. TIM-1 stimulation leads to increased B7-1, B7-H1, and PD-L2 expression, while inhibiting B7-H2 expression. These studies suggest that TIM-1 can regulate macrophage activation and alter the co-stimulatory properties of macrophages and thus may contribute to allergic inflammatory diseases such as asthma.     

Up-regulation of growth factor independence 1 in endotoxin tolerant macrophages with low secretion of TNF-α and IL-6

Objective

Endotoxin tolerance refers to a low response to lipopolysaccharide (LPS). We hypothesized that growth factor independence 1 (Gfi1) involves in the endotoxin tolerance in macrophages.

Methods

Endotoxin tolerance was induced in the RAW264.7 cell line and thioglycolate-elicited murine peritoneal macrophages by incubation with 100 ng/ml LPS for 20 h. Macrophages without the pretreatment were set as control. Both endotoxin tolerant and control cells were then stimulated with 1,000 ng/ml LPS for indicated period of incubation. Gfi1 mRNA expression and protein production were investigated by real-time PCR and Western blotting, respectively. ELISA was performed to quantify the secretion of TNF-α and IL-6.

Result

Compared with non-endotoxin tolerant macrophages, endotoxin tolerant cells secreted a lower amount of TNF-α and IL-6. The mRNA expression of Gfi1 in endotoxin tolerant macrophages was higher than that of control in both RAW264.7 cells and thioglycolate-elicited murine peritoneal macrophages. The protein production was accordingly up-regulated in endotoxin tolerant RAW264.7 cells.

Conclusion

In in vitro endotoxin tolerant macrophages, the expression of Gfi1 mRNA and protein were up-regulated after high dose LPS stimulation, accompanied with a blunted TNF-α and IL-6 secretion. Gfi1 might participate in the mechanism of endotoxin tolerance.