SCARF1 在人THP-1 单核源性巨噬细胞抗烟曲霉菌刺激中的作用

目的:运用干扰腺病毒沉默THP1细胞中SCARF1基因研究其在体外抗烟曲霉中的作用。方法:用灭活的烟曲霉分生孢子(1×105 CFU/m L)于不同时间点处理THP-1细胞,RT-PCR分别检测SCARF1和TNF-αm RNA的表达;将Ad-si RNA-SCARF1转导细胞24 h后给予烟曲霉孢子刺激24 h,通过RT-PCR法检测细胞中TNF-αm RNA表达,Western blot法检测细胞中SCARF1表达以及NF-κB通路相关信号分子的活性。结果:RT-PCR证实烟曲霉孢子刺激能时间依赖性增强THP1细胞中SCARF1和TNF-α表达;Western法证实与Ad-GFP组比较Ad-si RNA-SCARF1组SCARF1的表达量显著降低(P0.05),沉默效率为71%;与Ad-GFP组比较,Ad-GFP+Af组NF-κB亚单位p65的磷酸化水平明显升高(P0.05),在Ad-si RNA-SCARF1+Af组,磷酸化p65的产生明显减少,SCARF1沉默后细胞因子TNF-α的分泌明显减少。结论:烟曲霉孢子刺激能诱导巨噬细胞SCARF1的表达增加,诱导信号分子NF-κB的活化,导致相应的细胞因子分泌增加,从而在巨噬细胞抗烟曲霉中发挥作用。     

肾上腺素对THP-1巨噬细胞TGF-β1、SR-A1和CD36表达的影响

目的:探讨肾上腺素对THP-1巨噬细胞TGF-β1、SR-A1和CD36表达的影响。方法:不同浓度肾上腺素处理THP-1巨噬细胞24h,运用逆转录多聚酶链反应检测其TGF-β1、SR-A1和CD36的mRNA表达,酶联免疫吸附试验检测TGF-β1蛋白的表达,Westem-blotting检测SR-A1蛋白的表达。结果:100nmol/L及1μmol/L的肾上腺素作用THP-1巨噬细胞,引起TGF-β1 mR- NA和蛋白表达下调(p<0.05),SR-A1 mRNA和蛋白表达上调(P<0.05),1pmol/L～1μmol/L的肾上腺素处理后,各组CD36 mRNA水平无显著性变化(p>0.05)。结论:应激浓度的肾上腺素可能通过影响TGF-β1和SR-A1的表达而参与和/或促进AS的发生发展。     

普伐他汀联合罗格列酮上调THP-1巨噬细胞ABCA1表达

目的：观察普伐他汀与罗格列酮联合应用对人巨噬细胞株（THP-1）源性巨噬细胞三磷酸腺苷结合盒转运体A1（ABCA1）表达的影响。方法：THP-1细胞经160 nmol/L佛波酯（PMA）孵育24 h,诱导分化成巨噬细胞,分别与普伐他汀及罗格列酮单独或联合作用24 h,提取各组细胞总RNA和蛋白质,分别采用RT-PCR和Western blot检测ABCA1的mRNA和蛋白的表达。结果：普伐他汀增强过氧化物酶体增殖物激活受体γ（PPARγ）mRNA表达,但抑制肝X受体（LXR）mRNA表达（P〈0.05）,对ABCA1的表达不产生明显效应（P〉0.05）;罗格列酮单独或与普伐他汀联合作用均可引起ABCA1表达明显增加,同时PPARγ及LXRαmRNA表达亦上调（P〈0.05））。结论：普伐他汀与罗格列酮联合应用能上调巨噬细胞ABCA1的表达。     

鸟分枝杆菌PPE25蛋白对THP-1巨噬细胞免疫效应的影响

[目的]探讨鸟分枝杆菌PPE25在调控人源THP-1巨噬细胞免疫应答反应中的作用及机制.[方法]通过基因扩增、载体构建、诱导表达、分离纯化等过程制备鸟分枝杆菌重组PPE25蛋白;将重组PPE25蛋白作用于THP-1巨噬细胞后,利用流式细胞术检测巨噬细胞凋亡率;运用ELISA方法检测细胞上清中TNF-α的浓度;采取Wes...     

脓肿分枝杆菌感染THP-1巨噬细胞自噬相关分子的识别

为了探索THP-1巨噬细胞在脓肿分枝杆菌(Mycobacterium abscessus, MAB)感染后,与自噬相关的关键分子及其介导的生物学过程,从GEO和GeneCards数据库中挖掘MAB感染后THP-1巨噬细胞的自噬相关基因;利用DAVID在线工具对筛选的差异自噬相关基因进行基因本体论(GO)、京都基因和基因组数据库(KEGG)富集分析;采用实时荧光定量PCR (qRT-PCR)方法验证筛选的关键基因;将关键基因与自噬标志分子MAP1LC3A进行Pearson相关性分析。生物信息学分析显示,与自噬密切相关的差异表达基因共11个,其中前5个是BNIP3 (Bcl-2/adenovirus E1 B interacting protein 3)、BAMBI (BMP and activin membrane bound inhibitor)、MAP1LC3A (microtubule associated protein 1 light chain 3)、DYSF (Dysferlin)和MYOZ1 (Myozenin1);功能预测显示, 11个差异表达基因主要和自噬等过程相关;...     

干扰素IFNL4对巨噬细胞系THP-1免疫应答的影响

目的:分析Interferon-lambda 4(IFNL4)表达与巨噬细胞免疫应答间的关系,探讨IFNL4调控免疫应答的信号机制,发掘IFNL4在免疫调理方面的潜在应用价值。方法:建立THP-1细胞培养及分化刺激体系,使用RT-PCR检测不同分化状态THP-1细胞IFNL4的表达水平,并在THP-1细胞中过表达IFNL4,检测IFNL4过表达对THP-1细胞分泌IL-12、TNF-α、IL-10和TGF-β等细胞因子及细胞迁移效率的影响。结果:THP-1细胞分化抑制IFNL4的表达,分化前比分化后IFNL4表达水平相对定量下降255.46倍,差异显著性P0.001;M2极化巨噬细胞较M1细胞表达IFNL4因子水平升高14.69倍,显著性差异P=0.009;IFNL4过表达可抑制IL-12和TNF-α表达水平,其中TNF-α表达水平变化具有统计学意义(P=0.017),表达水下降5.97倍。IFNL4可促进IL-10和TGF-β的表达,其中TGF-β变化具有统计学意义(P=0.046),表达水平相对上升2.42倍。且IFNL4对THP-1细胞迁移效率具有抑制作用(P=0.005),刺激前细胞迁移数为45.33,IFNL4刺激后迁移数为32.67,迁移移效率下降1.39倍。结论:干扰素IFNL4在分化的M2型THP-1巨噬细胞中具有较高的表达水平,且对THP-1的免疫应答具有一定的抑制作用。     

转化生长因子-β1对前单核白血病细胞系THP-1的分化诱导作用

本文研究了重组人转化生长因子-β1对前单核白血病细胞系THP-1细胞增殖抑制作用和分化诱导作用。细胞染色计数和~3H-TdR掺入数据表明,在0.078—1.25 ng/ml浓度范围内,rhTGF-β1剂量与增殖抑制呈正相关。在抑制细胞增殖的同时,rhTGF-β1诱导细胞分化,诱导体系中细胞形态和生长方式均发生明显变化,细胞转为贴壁生长,表现出Mφ的细胞形态。α-醋酸萘酚酯酶、硝基蓝四唑还原、细胞吞噬试验均表明细胞具有Mφ的生物功能。电镜观察显示分化细胞具有典型的巨噬细胞样细胞形态,胞内出现Mφ所特有的亚细胞结构初级溶酶体和次级溶酶体。另外,TGF-β特异性中和单抗TB 21对实验体系THP-1细胞分化的阻断证明,THP-1细胞的分化确为rhTGF-β1诱导所致。     

氧化低密度脂蛋白对THP-1巨噬细胞吞噬和分泌功能的影响

目的:探讨氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞源性泡沫细胞吞噬功能和炎症相关因子分泌功能的影响。方法:利用佛波酯(phorbol ester,PMA)诱导THP-1细胞分化形成巨噬细胞,之后采用ox-LDL处理48小时后,诱导其形成泡沫细胞。利用中性红吞噬实验,分析泡沫细胞形成前后吞噬功能的变化;通过ELISA法,检测细胞培养上清中肿瘤坏死因子α(tumor necrosis factorα,TNF-α)含量,观察ox-LDL对THP-1巨噬细胞功能的影响。结果:细胞形态学结果表明,我们成功利用ox-LDL诱导THP-1巨噬细胞形成泡沫细胞;进一步发现ox-LDL诱导THP-1巨噬细胞表面的清道夫受体CD36表达升高,并促进细胞吞噬功能增加,进一步促进细胞内胆固醇含量显著升高(P0.05);同时,ox-LDL能够刺激巨噬细胞大量分泌TNF-α(P0.05)。结论:ox-LDL通过增强清道夫受体CD36表达,提高巨噬细胞的吞噬功能,引起大量胆固醇聚集,产生细胞毒性损伤,并促进TNF-α炎性因子的大量分泌。     

The effect of myeloperoxidase-oxidized LDL on THP-1 macrophage polarization and repolarization

Macrophages (Mφs) play a crucial role in the development of atherosclerosis by engulfing modified LDL particles and forming foam cells, the hallmark of atherosclerosis. Many studies suggest that myeloperoxidase-oxidized LDL (Mox-LDL) is an important pathophysiological model for LDL modification in vivo. Classically (M1) and alternatively activated (M2) Mφs are both implicated in the process of atherogenesis. Mφs are highly plastic cells whereby they undergo repolarization from M1 to M2 and vice versa. Since little is known about the effects of Mox-LDL on Mφ polarization and repolarization, our study aimed at evaluating the in vitro effects of Mox-LDL at this level through making use of the well-established model of human THP-1-derived Mφs. Resting M0-Mφs were polarized toward M1- and M2-Mφs, then M0-, M1- and M2-Mφs were all treated with physiological concentrations of Mox-LDL to assess the effect of Mox-LDL treatment on Mφ polarization and repolarization. Treatment of M0-Mφs with a physiological concentration of Mox-LDL had no significant effects at the level of their polarization. However, treatment of M1-Mφs with Mox-LDL resulted in a significant reduction in their IL-10 cytokine secretion. Our results point to a potential role of Mox-LDL in increasing the pro-inflammatory state in Mφs through reducing the release of the anti-inflammatory cytokine, IL-10.     

Punicalagin Targets Atherosclerosis: Gene Expression Profiling of THP-1 Macrophages Treated with Punicalagin and Molecular Docking

Atherosclerosis is an important cause of cardiovascular disorders worldwide. Natural botanical drugs have attracted attention due to their antioxidant, anti-inflammatory, and antiatherogenic properties in the treatment of atherosclerosis. Punicalagin is the major bioactive component of pomegranate peel, and has been shown to have antioxidant, anti-inflammatory, antiviral, anti proliferation, and anticancer properties. To explore its antiatherogenic properties at a molecular level, we investigated the genome-wide expression changes that occur in differentiated THP1 cells following treatment with a non-toxic dose of punicalagin. We also conducted a molecular docking simulation study to identify the molecular targets of punicalagin.     

Metformin induced expression of Hsp60 in human THP-1 monocyte cells

Metformin is in widespread clinical use for the treatment of diabetes mellitus in patients. It has been shown to inhibit mitochondrial bioenergetic functions by inhibiting complex I of the electron transport chain. The expression of mitochondrial-specific molecular stress protein Hsp60 is a key consequence of mitochondrial impairment. Since this protein has important immune-modulatory properties, we have investigated the expression of Hsp60 in human THP-1 monocyte cells exposed to metformin. In this study, we demonstrate significant up-regulation of Hsp60 at both mRNA and protein levels when these cells were exposed to metformin at therapeutic dosage levels. Interestingly, there was also an increase in expression of CD14 mRNA in these cells. This suggested a possible modulation of the differentiation rates of the THP-1 cells during exposure to metformin. As monocyte differentiation marks a critical step in atherosclerosis, these observations suggest that long-term exposure to metformin could have important implications for the diabetic patient.     

ApoAⅠ促进THP-1源性泡沫细胞ApoE分泌的研究

目的通过建立动脉粥样硬化损伤部位细胞模型,观察ApoAI对THP—l源性泡沫细胞ApoE分泌的影响,探讨ApoAI与ApoE之间是否存在相互关系以及这种关系对动脉粥样硬化的影响。方法采用聚乙二醇-6000（PEG-6000）沉淀法制备人血浆HDL,再利用凝胶过滤柱层析法分离ApoA I;佛波酯（PMA）诱导THP-1单核细胞分化为巨噬细胞,氧化型低密度脂蛋白（oxidized low density lipoprotein,OXLDL）使分化后THP-1细胞荷脂,建立动脉粥样硬化细胞模型;ELISA检测不同浓度ApoAI（0,5,10,15,25ug／m1）及不同孵育时间（0．3,6,12,24h）,THP—1源性泡沫细胞ApoE的分泌;RT—PCR检测细胞ApoEmRNA的表达情况。结果ApoAI增加THP-1源性泡沫细胞ApoE的分泌量;且ApoE蛋白质的分泌随着ApoAI孵育时间增加而增加,在24hApoE的分泌量达最大;在剂量试验中,ApoE的分泌量随着ApoAI剂量的增加有增加趋势,ApoAI浓度为25μmol／L时,ApoE分泌量最大;而却oE基因的表达不受ApoAI的影响。结论-定浓度的ApoAI促进THP-1源性泡沫细胞ApoE的分泌,且具有时间及剂量依赖性。而在基因水平上,ApoAI对ApoEmRNA表达没有影响。     

Redistribution of macrophage cholesteryl ester hydrolase from cytoplasm to lipid droplets upon lipid loading

Hydrolysis of intracellular cholesteryl esters (CEs) represents the first step in the removal of cholesterol from lipid-laden foam cells associated with atherosclerotic lesions. Neutral cholesteryl ester hydrolase (CEH) catalyzes this reaction, and we recently cloned the cDNA for the human macrophage CEH and demonstrated increased mobilization of intracellular CE droplets by CEH overexpression. The present study was undertaken to test the hypothesis that for CE hydrolysis, CEH must become associated with the surface of the cytoplasmic lipid droplets. Our data show the redistribution of CEH from cytosol to lipid droplets upon lipid loading of human THP-1 macrophages. Depletion of triacylglycerol (TG) by incubation with the acyl-CoA synthetase inhibitor Triacsin D had no effect on CEH association with the lipid droplets, suggesting that CEH associates with mixed (CE + TG) as well as TG-depleted CE droplets. However, CEH had 2.5-fold higher activity when mixed droplets were used as substrate in an in vitro assay, consistent with the reported higher cholesterol efflux from cells containing mixed isotropic droplets. Perilipin as well as adipophilin, two lipid droplet-associated proteins, were also present on the lipid droplets in THP-1 macrophages. In conclusion, CEH associates with its intracellular substrate (lipid droplets) and hydrolyzes CE more efficiently from mixed droplets.