肺炎嗜衣原体CPAF诱导THP-1细胞产生前炎症细胞因子和凋亡

目的 研究肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)衣原体蛋白酶样活性因子(Chlamydial protease-like activity factor,CPAF)能否在体外诱导人单核细胞THP-1产生前炎症细胞因子和凋亡,为进一步探索Cpn感染宿主致病的分子机制提供实验依据.方法 将Cpn CPAF全基因克隆于pGEX6p-2载体上,在大肠杆菌(E coli)中诱导表达重组蛋白,经去内毒素纯化柱和琼脂糖凝胶FF获得纯化且无脂多糖(LPS)污染的重组蛋白GST-CPAF.以不同浓度的该蛋白作用于THP-1,ELISA检测TNF-α吨和IL-6水平.MTT法检测经该蛋白处理后THP-1的增生或抑制作用,用Hoechst33258荧光染色、DNA片段化分析、Armexin V-FITC-PI染色法检测细胞凋亡情况.结果 制备的莺组蛋白GST-CPAF以时间和剂量依赖方式刺激THP-1分泌TNF-α和IL-6,并以剂量依赖方式抑制THP-1增殖;当GST-CPAF刺激THP-1细胞24 h后,能诱导细胞调亡.结论 制备的Cpn重组蛋白GST-CPAF能诱导THP-1产生前炎症细胞因子和凋亡,可能是Cpn致病机制中的一个因素.     

钙离子对重组创伤弧菌溶细胞素诱导THP-1细胞损伤的影响机制

目的 研究重组创伤弧菌溶细胞素( rVvhA)诱导人单核细胞白血病细胞(THP-1)的凋亡机制及其Ca2+的变化.方法 采用CCK-8法、激光共聚焦显微镜结合Fluo 3/AM法、流式细胞术结合AnnexinV -PI标记等检测rVvhA对THP-1细胞的影响,并观察胞内Ca2+浓度变化.结 果rVvhA可诱导THP-1细胞发生凋亡并引起细胞内Ca2+浓度升高,细胞内钙离子螯合剂BAPTA-AM处理组胞内钙离子升高幅度远高于细胞外钙离子螯合剂EGTA处理组.结论 rVvhA具有诱导THP-1细胞凋亡的生物学活性,并能引起细胞内Ca2+浓度升高,升高的Ca2+主要源于胞外钙离子内流.     

HIV-1 Nef基因在THP-1细胞的表达和活性检测

目的将HIV-1 Nef基因转染THP-1细胞,获得稳定表达Nef蛋白的细胞克隆,为研究Nef对巨噬细胞生物学活性的影响奠定实验基础。方法将质粒pcDNA3.1(+)-Nef和pcDNA3.1(+()阴性对照)转染THP-1细胞,通过逆转录-聚合酶链反应(RT-PCR)、Western blot、细胞免疫荧光等方法检测目的蛋白在真核细胞内的表达及定位,采用共转染法将荧光报告基因转染THP-1Nef和THP-13.1细胞,通过测定荧光(RLU)值来评价Nef蛋白的生物学活性。结果转染细胞经G418筛选后获得稳定表达Nef的THP-1细胞株,RT-PCR及Western blot结果表明Nef在真核细胞中成功表达,细胞免疫荧光结果显示,THP-1-Nef细胞表达的Nef蛋白主要定位于细胞质中。荧光酶标仪检测转染了HIV-1LTR-Luc和NFκB-Luc荧光报告基因的THP-1-Nef和THP-1-3.1细胞的RLU值。结论成功建立了THP-1-Nef细胞稳定表达细胞株,检测了其生物学活性,为进一步研究其作用机制实验奠定基础。     

NPM1基因突变表达对THP-1白血病细胞增殖和凋亡的作用探讨

 摘要：目的 探讨核仁磷酸蛋白（NPM1）基因突变对白血病细胞增殖潜能和凋亡发生的影响。方法 将携带NPM1 A型突变（NPM1 mA）的重组质粒载体pEGFPC1-NPM1 mA转染白血病THP-1细胞系,构建稳定表达NPM1 A型突变蛋白的白血病细胞株(THP-1 mA),同时设立未处理组（THP-1）和空载体转染组（THP-1 C1）作为对照。通过MTT试验观察细胞体外增殖能力,流式细胞术分析细胞周期分布及凋亡发生率的改变;采用RT-PCR和Western blot分别检测细胞凋亡相关蛋白（Bax和Bcl-2）mRNA及蛋白表达水平。结果 与未处理组和空载体转染组细胞相比较,携NPM1突变体的THP-1 mA细胞体外增殖能力明显增强,S期细胞比例明显增高,G1期细胞比例显著减低;而NPM1突变体转染后THP-1细胞的凋亡率没有显著变化,同时细胞凋亡相关蛋白Bax, Bcl-2的mRNA和蛋白表达以及Bax/Bcl-2比值亦未见明显改变。结论 NPM1突变基因能够促进白血病细胞的体外增殖能力,而对白血病细胞凋亡无显著影响,这为进一步阐明NPM1突变与AML的关系提供了新的科学依据。     

熊果酸对THP-1细胞HMGB1表达和NF-kB活性的影响

目的 研究熊果酸(UA)对人单核细胞系THP-1细胞HMGB1表达和NF-kB活性的影响.方法 用不同浓度的UA(0、0.1、1、5和10 μmol/L)作用细胞后进行如下检测:1)MTr检测细胞增殖;2)RT-PCR法检测HMGB1和P65 mRNA表达;3)Western blot检测HMGB1和P65蛋白的表达;4)荧光素酶报告基因转染,质粒pNF-kB-luc瞬时转染THP-1细胞,转染40h后加1μmol/L UA作用,于转染48 h后进行荧光素酶活性测定.结果 1)随着UA浓度的增高,可明显抑制THP-1细胞的增殖;2)不同浓度的UA对THP-1细胞HMGBl mRNA和蛋白表达,呈现出一定的双向性,且以l μmol/L UA作用最强(P ＜0.05);3)UA可增强P65 mRNA和蛋白表达以及NF-kB活性.结论 UA可影响单核细胞HMGB1表达和NF-kB活性.     

Serum levels of reactive oxygen species (ROS) in the bitch

Abstract

The aim of this study was to determine the serum concentrations of reactive oxygen species (ROS) during the different phases of the estrous cycle in the bitch, in order to establish their physiological values. 56 healthy mixed-breed bitches were enrolled at this purpose and divided into 4 groups, standing on the different phases of the estrus cycle. Blood samples were collected in all groups and serum ROS concentrations were determined. Proestral concentrations were statistically higher than anestral ones, and statistically lower than those found in estrus (p<0.001). The highest concentrations of ROS were detected at estrus, that is, in the peri-ovulatory period. This sharp increase in ROS concentrations is related to the acute inflammatory process underlying ovulation and to the increase in immune and metabolic activities, cytological changes and myometrial contractility promoted by the high levels of estrogens. In diestrus, the mean concentration of ROS decreases. This reduction did not show any statistically significant difference with the mean value observed in proestrus. In this phase, in fact, the high concentrations of progesterone, exerting an antioxidant and immunodepressive effect, justify the lower mean concentration of ROS detected. In anestrus, the lowest concentrations of ROS were observed, for the reduced metabolic and endocrine activity occurring in this phase of the estrous cycle. In conclusion our results establish the physiologic levels of ROS during the estrous cycle in the bitch and reflect the endocrine morphologic and metabolic changes occurring during it.     

Multitalented EspB of enteropathogenic Escherichia coli (EPEC) enters cells autonomously and induces programmed cell death in human monocytic THP-1 cells

Enteropathogenic Escherichia coli (EPEC) subvert host cell signaling pathways by injecting effector proteins via a Type 3 Secretion System (T3SS). The T3SS-dependent EspB protein is a multi-functional effector protein, which contributes to adherence and translocator pore formation and after injection exhibits several intracellular activities. In addition, EspB is also secreted into the environment. Effects of secreted EspB have not been reported thus far. As a surrogate for secreted EspB we employed recombinant EspB (rEspB) derived from the prototype EPEC strain E2348/69 and investigated the interactions of the purified protein with different human epithelial and immune cells including monocytic THP-1 cells, macrophages, dendritic cells, U-937, epithelial T84, Caco-2, and HeLa cells. To assess whether these proteins might exert a cytotoxic effect we monitored the release of lactate dehydrogenase (LDH) as well as propidium iodide (PI) uptake. For comparison, we also investigated several homologs of EspB such as IpaD of Shigella, and SipC, SipD, SseB, and SseD of Salmonella as purified recombinant proteins. Interestingly, cytotoxicity was only observed in THP-1 cells and macrophages, whereas epithelial cells remained unaffected. Cell fractionation and immune fluorescence experiments showed that rEspB enters cells autonomously, which suggests that EspB might qualify as a novel cell-penetrating effector protein (CPE). Using specific organelle tracers and inhibitors of signaling pathways we found that rEspB destroys the mitochondrial membrane potential – an indication of programmed cell death induction in THP-1 cells. Here we show that EspB not only constitutes an essential part of the T3SS-nanomachine and contributes to the arsenal of injected effector proteins but, furthermore, that secreted (recombinant) EspB autonomously enters host cells and selectively induces cell death in immune cells.     

Neurokinin-1 receptor activation induces reactive oxygen species and epithelial damage in allergic airway inflammation

Background An induction of reactive oxygen species (ROS) is characteristic for inflammation but the exact pathways have not been identified for allergic airway diseases so far. Objective The aim of this study was to characterize the role of the tachykinin NK‐1 receptor on ROS production during allergen challenge and subsequent inflammation and remodelling. Methods Precision‐cut lung slices of ovalbumin (OVA)‐sensitized mice were cultivated and ROS‐generation in response to OVA challenge (10 μg/mL) was examined by the 2′,7′‐dichloroflourescein‐diacetate method. Long‐term ROS effects on epithelial proliferation were investigated by 5‐bromo‐2′‐deoxyuridine incorporation (72 h). In vivo, the results were validated in OVA‐sensitized animals which were treated intra‐nasally with either placebo, the tachykinin neurokinin 1 (NK‐1) receptor antagonist SR 140333 or the anti‐oxidant N‐acetylcystein (NAC) before allergen challenge. Inflammatory infiltration and remodelling were assessed 48 h after allergen challenge. Results ROS generation was increased by 3.7‐fold, which was inhibited by SR 140333. [Sar⁹,Met¹¹(O₂)]‐Substance P (5 nm ) caused a tachykinin NK‐1 receptor‐dependent fourfold increase in ROS generation. Epithelial proliferation was decreased by 68% by incubation with [Sar⁹,Met¹¹(O₂)]‐SP over 72 h. In‐vivo, treatment with SR 140333 and NAC reduced epithelial damage (91.4% and 76.8% vs. placebo, respectively, P<0.01) and goblet cell hyperplasia (67.4% and 50.1% vs. placebo, respectively, P<0.05), and decreased inflammatory cell influx (65.3% and 45.3% vs. placebo, respectively, P<0.01). Conclusion Allergen challenge induces ROS in a tachykinin NK‐1 receptor‐dependent manner. Inhibition of the tachykinin NK‐1 receptor reduces epithelial damage and subsequent remodelling in vivo. Therefore, patients may possibly benefit from treatment regime that includes radical scavengers or tachykinin NK‐1 receptor antagonists.     

Characterization of entry and infection of monocytic THP-1 cells by Kaposi's sarcoma associated herpesvirus (KSHV): Role of heparan sulfate, DC-SIGN, integrins and signaling

KSHV effectively binds, enters and establishes infection in THP-1 cells with initial concurrent expression of latent ORF73 and lytic ORF50 genes and subsequent persistence of ORF73. KSHV genome persisted for 30 days and lytic cycle could be activated. KSHV utilized heparan sulfate for binding to THP-1 cells and primary monocytes. Blocking DC-SIGN did not inhibit KSHV binding; however, virus entry in THP-1 cells and in primary monocytes was reduced. In addition to the previously identified integrins α3β1, αvβ3 and αvβ5, integrin α5β1 was also utilized for infection. KSHV entered THP-1 cells via clathrin and caveolin mediated endocytosis and did not utilize macropinocytosis as in human dermal endothelial cells, and required an endosomal acidification. Infection also induced phosphorylation of FAK, Src, PI3K, NF-κB and ERK1/2 signaling molecules, and entry was blocked by tyrosine kinase inhibitors. These findings suggest that THP-1 cells are highly useful model for studying KSHV infection of monocytes.     

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin‐1 beta (IL‐1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL‐1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL‐1β processing in human neutrophils is dependent on caspase‐1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase‐1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL‐1β; whereas ROS negatively controlled caspase‐1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL‐1β out of the cell, a role never previously described. The complex ROS‐mediated regulation of neutrophil IL‐1β secretion might constitute a physiological mechanism to control IL‐1β‐dependent inflammatory processes where neutrophils play a crucial role.     

人单核细胞系THP-1中HMGN2的分离纯化及其抗菌活性

 目的 从人单核细胞株THP-1中分离与纯化HMGN2抗菌肽,应用微量琼脂糖弥散法检测抗菌活性,以探讨HMGN2分子的抗菌作用。方法 采用HPLC分离纯化, 琼脂糖弥散法筛选纯化有抗菌活性的组分,用Tricine-SDS-PAGE电泳初步测定其分子质量,以质谱法进行分子质量的精确分析。结果 从人THP-1细胞中分离纯化出HMGN2抗菌肽, 琼脂糖弥散法检测显示其对大肠杆菌氨苄青霉素耐药株ML-35p有较强的抑菌活性,对大肠杆菌标准株ATCC25922和临床分离株54080亦有较强的抗菌活性,而对金黄色葡萄球菌ATCC25923未检测出抗菌活性。结论：HMGN2是一种抗菌分子,可能参与了体内的天然免疫防御作用。     

From death receptor to reactive oxygen species and c-Jun N-terminal protein kinase: the receptor-interacting protein 1 odyssey

Summary: Death receptors (DRs) are more than simple killers: they control cell growth, proliferation, and survival, thereby playing a pivotal role in immune and inflammatory responses. Some of these phenomena might be explained by aberrant reactive oxygen species (ROS) production and metabolism, which can lead to oxidative stress. A key signaling molecule of DR-initiated intracellular pathways, receptor-interacting protein 1 (RIP1), orchestrates a complex control of multiple responses and may link DR-associated signaling complexes to ROS production by mitochondria. Yet, RIP1 is also an important regulator of endogenous anti-oxidants and ROS scavenging enzymes, because it is required for nuclear factor κB activation that results in expression of anti-apoptotic and anti-oxidant proteins. Alteration of RIP1 function may result in ROS accumulation and abnormal c-Jun N-terminal protein kinase activation, affecting inflammatory responses, innate immunity, stress responses, and cell survival. These molecular mechanisms may be involved in neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases.     

Modulation of IL-37 expression by triptolide and triptonide in THP-1 cells

IL-37 is an anti-inflammatory cytokine that was only recently identified, and it is highly expressed in tissues from patients with inflammatory and autoimmune diseases. Inflammatory cytokines and inflammatory stimuli can induce the upregulation of IL-37. However, it has not been reported whether anti-inflammatory medications induce the expression of IL-37. In this work, we uncovered, for the first time, that two main bioactive components, triptolide and triptonide, from the herb Tripterygium wilfordii Hook f. (TwHF), which possess anti-inflammatory activity, upregulate the expression of IL-37, and this expression was suppressed by ERK1/2 and p38 MAPK inhibitors. Overall, our research demonstrated, for the first time, that anti-inflammatory active components (triptolide and triptonide) upregulated the expression of IL-37 most likely via activation of the ERK1/2 and p38 MAPK pathways.     

启动子区域甲基化状态对THP-1细胞肿瘤坏死因子α分泌的影响

目的: 探讨肿瘤坏死因子α( TNF-α )基因启动子区域散在CpG双核苷酸结构甲基化状态对蛋白分泌和基因表达的影响。方法: 检测人单核细胞系THP-1细胞在脂多糖(LPS)刺激状态下,不同时点TNF-α的表达和其基因启动子区域的甲基化状态。采用ELISA法测定细胞培养液上清TNF-α水平;采用重亚硫酸盐转化基因测序法测定DNA甲基化状态。结果: 受到LPS刺激后,THP-1细胞迅速产生 TNF-α ,在4 h达到高峰后快速下降。 TNF-α 基因启动子-344 bp到转录起始位点(TSS)区域内,存在11个散在CpG双核苷酸结构。未受到LPS刺激时均处于甲基化状态;刺激4 h后,有4个处于甲基化状态;刺激8 h后,有9个处于甲基化状态。未刺激与刺激4 h之间,刺激4 h与刺激8 h之间,甲基化率比较均有显著差异(P＜0.01,P<0.05)。结论: 未受到LPS刺激时,THP-1细胞 TNF-α 基因启动子区域甲基化水平较高;LPS刺激后,其该区域甲基化水平降低。该变化与TNF-α表达分泌相关。     

溶血磷脂酸对基质金属蛋白酶-9表达及活性的影响

目的观察溶血磷脂酸（Lysophosphatidie Acid,LPA）对人单核细胞株THP-1细胞基质金属蛋白酶-9（matrix metalloproteinases-9）表达及活性和NF-κB p65表达活性的影响,探讨MMP-9在LPA致动脉粥样硬化中的作用及机制。方法以不同浓度水平LPA（0-10μmol/L）刺激人THP-1细胞4h,RT-PCR法测定MMP-9mRNA表达,酶谱法检测MMP-9活性,westernblot检测核蛋白NF-κB p65表达变化。结果随着LPA剂量的增加,MMP-9表达及活性,核蛋白NF-κB p65表达逐渐增强,在LPA1μmol/L时达到最高点,然后逐渐减弱,LPA以剂量依赖的方式激活NF-κB,上调THP-1细胞MMP-9表达,增加MMP-9活性。结论LPA可能通过激活NF-κB,在转录水平促进THP-1细胞MMP-9mRNA表达,并增强其活性,促进粥样硬化发生和发展。     

Mycophenolic acid enhanced lipopolysaccharide-induced interleukin-18 release in THP-1 cells via activation of the NLRP3 inflammasome

Abstract

Background: Interleukin (IL)-18 is a pro-inflammatory cytokine that has important functions in host defense. The maturation and secretion of IL-18 has been shown to be regulated by the NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome. Mycophenolic acid (MPA), the active metabolite of mycophenolate mofetil (MMF), in association with lipopolysaccharide (LPS), is able to promote the secretion of IL-18, but the mechanism remains unknown. This study aims to explore the mechanism by which MPA synergizes with LPS to induced IL-18 release.

Methods: THP-1 cells were stimulated with LPS and MPA and treated with or without the inhibitors of caspase-1, Ac-YVAD-cmk or KCl; IL-18 in the supernatants was measured by ELISA. The intracellular protein levels of NF-κB p-p65, pro-IL-18, NLRP3, and cleaved caspase-1(p20) were measured by Western blot.

Results: We found that MPA alone failed to induce IL-18, whereas MPA enhanced LPS-mediated IL-18 release. MPA did not affect the intracellular protein levels of NF-κB p-p65 or pro-IL-18 but activated the NLRP3 inflammasome. Ac-YVAD-cmk or increasing extracellular K⁺ blocked the activation of caspase-1 and attenuated the release of IL-18.

Conclusions: Taken together, MPA synergized with LPS to induce the release of IL-18 via activating the NLRP3 inflammasome and increasing the degradation of pro-IL-18, rather than by enhancing the production of pro-IL-18.     

转化生长因子－β1诱导前单核自血病细胞分化过程中细胞表型变化的研究

本文对重组人转化生长因子－β１诱导前单核白血病细胞系ＴＨＰ－１细胞向ＭΦ分化过程中细胞表面特异性抗原表达变化进行了研究。流式细胞仪分析表明，实验细胞ＴＨＰ－１主要表现单核细胞的表型，巨噬细胞标志Ｍａｃ－１α、Ｍａｃ－１β、Ｍａｃ－２为阴性。ｒｈＴＧＦ－β１作用后，分化细胞表面单核细胞标志４Ｆ２表达下降，巨噬细胞特异抗原Ｍａｏ－１α、Ｍａｃ－１β、Ｍａｃ－２表达上升，细胞表现出ＭΦ表型。结合前文分化细胞表现出的脱氢酶硝基蓝四唑还原、α－萘酚醋酸酯酶活性和吞噬功能，可以发现分化细胞在细胞表面特异性抗原表达及细胞表型的转变与其ＭΦ生物学功能的获得在时相上基本一致。