不对称二甲基精氨酸对单核/巨噬细胞分化为泡沫细胞过程中酰基辅酶A胆固醇酰基转移酶-1表达的影响

 [摘要] 目的 观察不对称二甲基精氨酸（ADMA）对单核/巨噬细胞分化为泡沫细胞过程中酰基辅酶A胆固醇酰基转移酶-1（ACAT-1）表达的影响。 方法 （1）THP-1单核细胞与160nmol/L佛波酯（PMA）孵育48h后,再与80mg/L氧化型低密度脂蛋白（ox-LDL）孵育24h,用油红O染色在光镜下鉴定细胞形态及变化。（2） 将THP-1单核细胞、巨噬细胞、泡沫细胞分别用20μmol/L ADMA干预24h,酶法测泡沫细胞内胆固醇酯的含量,RT-PCR方法检测ACAT-1mRNA表达,Western blot方法检测其蛋白表达。 结果 （1）ADMA可显著增加泡沫细胞内胆固醇酯的含量。（2）与THP-1单核细胞相比,巨噬细胞（p<0.05）、泡沫细胞（p<0.01）中ACAT-1mRNA及蛋白表达增加;而与巨噬细胞相比,泡沫细胞中ACAT-1mRNA及蛋白表达增加但无显著性差异。（3）与各自的对照组相比,经ADMA作用后3种细胞中ACAT-1mRNA及蛋白表达水平均增加（p<0.01）。结论 ADMA增加泡沫细胞内胆固醇酯的含量可能是通过上调ACAT-1的mRNA及蛋白表达来实现的。     

MICA/B expression in macrophage foam cells infiltrating atherosclerotic plaques

Infiltrating macrophages accumulate in fatty streak lesions and transform into foam cells, leading to the formation of atherosclerotic plaques. Inflammatory mechanisms underlying the plaque formation mediated by NKG2D-positive lymphocytes such as CD8⁺ T cells, natural killer cells and natural killer T cells have been extensively investigated. Yet, the involvement of the NKG2D system itself remains poorly understood. Recent work in mouse models has shown that blockade of an NKG2D receptor–ligand interaction reduces plaque formation and suppresses inflammation in aortae. In this study, we conducted immunohistochemical analysis of NKG2D ligand expression in autopsy-derived aortic specimens. Foam cells expressing NKG2D ligands MICA/B were found in advanced atherosclerotic lesions accompanied by a large necrotic core or hemorrhage. Human monocyte-derived macrophages treated in vitro with acetylated low-density lipoproteins enhanced expression of MICA/B and scavenger receptor A, thus accounting for NKG2D ligand expression in foam cells infiltrating atherosclerotic plaques. Our results suggest that, as in mice, the NKG2D system might be involved in the development of atherosclerosis in humans.     

花青素对小鼠巨噬泡沫细胞胆固醇外流的影响

目的：研究植物化学物质花青素对小鼠巨噬泡沫细胞胆固醇外流的影响，探讨花青素促进巨噬泡沫细胞胆固醇外流的分子机制。方法： 制备乙酰化低密度脂蛋白（AcLDL），以其负载小鼠腹腔巨噬细胞形成巨噬泡沫细胞，观察不同浓度花青素Cy-3-G和Pn-3-G对巨噬泡沫细胞胆固醇外流的影响以及调节胆固醇外流有关的基因过氧化物酶体增殖物激活受体-γ（PPAR-γ）表达的情况。结果：AcLDL可促进胆固醇在巨噬细胞中大量蓄积，造成泡沫细胞的形成。花青素Cy-3-G和Pn-3-G能引起巨噬泡沫细胞内胆固醇的大量外流，并且增加PPAR-γ基因的表达。结论：花青素Cy-3-G和Pn-3-G促进胆固醇外流的作用与其增加PPAR-γ基因的表达有关。     

胰岛素对THP-1细胞脂蛋白脂酶 mRNA表达及细胞泡沫化的影响

目的 观察胰岛素在氧化低密度脂蛋白(ox-LDL)致人髓系白血病单核细胞株THP-1细胞泡沫化过程中的作用,并探讨其机理.方法 用不同浓度胰岛素及ox-LDL与THP-1细胞一起共同孵育,观察THP-1细胞脂蛋白脂酶(LPL) mRNA RT-PCR产物、非特异性脂酶染色THP-1细胞所得脂酶阳性细胞数、细胞大小变化、油红O染色所得含脂滴细胞数、细胞内胆固醇含量.结果 100 mU/L以上浓度胰岛素各组THP-1细胞LPL mRNA表达与ox-LDL对照组比较上调2倍;细胞周长、脂滴阳性细胞百分率及细胞内胆固醇含量均明显高于ox-LDL对照组(P＜0.05).结论 高浓度胰岛素在ox-LDL存在条件下有促进THP-1细胞向泡沫细胞转化的作用,其机理可能与细胞LPL mRNA表达上调有关.     

溶血卵磷脂对巨噬泡沫细胞胆固醇外流的影响

目的：探讨溶血卵磷脂（LPC）对巨噬泡沫细胞胆固醇外流的影响，为动脉粥样硬化（AS）的防治研究提供理论依据。方法：分离培养小鼠腹腔巨噬细胞，密度梯度超速离心法分离人血浆低密度脂蛋白（LDL），乙酰化后形成乙酰化低密度蛋白（AcLDL),用AcLDL负载巨噬细胞使之形成巨噬泡沫细胞模型。用酶学荧光法检测细胞胆固醇外流，用乳酸脱氢酶（LDH）检测试剂盒检测培养基中LDH活性来反映LPC的细胞毒性。结果：LPC处理巨噬泡沫细胞24h后，培养基中胆固醇含量明显高于对照组（1．7－4倍），细胞内胆固醇含量明显低于对照组。且LPC引起细胞胆固醇外流增加同时，培养基中LDH活性并没有明显差异。结论：在10－80μmol/L剂量范围内，LPC可以剂量依赖性地促进巨噬泡沫细胞胆固醇外流，且这一效应与LPC的细胞毒性之间没有关系。     

TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation from monocyte/macrophage lineage precursor cells

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. Recent evidence indicates that the TNF-related apoptosis-inducing ligand (TRAIL) of the TNF ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated T cells. We show in this work that TRAIL can induce osteoclast formation from human monocytes and murine RAW264.7 macrophages. We demonstrated that both cell models differentiate into osteoclast-like cells in the presence of TRAIL in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. The TRAIL-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. However, TRAIL-induced osteoclastogenesis is dependent on activation of NF-kappaB, ERK, and p38 MAP kinase. Thus, our data demonstrate that TRAIL induces osteoclast differentiation via direct engagement with the TRAIL death receptor through a signaling pathway distinct from apoptosis. Our results indicate that in addition to triggering apoptosis, TRAIL induces osteoclast differentiation. It provides a novel role for TRAIL in regulating osteoclast differentiation and in osteoimmunology.     

Effects of lithium and rubidium on the differentiation of mononuclear cells

Rubidium ions added at 0.5 mEq to liquid cultures of human mononuclear cells stimulated the differentiation of monocyte and particularly of granulocyte cell lines.     

氧化低密度脂蛋白及溶血卵磷脂对巨噬泡沫细胞胆固醇外流的影响

目的:观察氧化低密度脂蛋白(OxLDL)及其成分溶血卵磷脂(LPC)对小鼠腹腔巨噬泡沫细胞胆固醇外流的影响及其机制的初步探讨。方法:(1)以载脂蛋白AI(apoAI)分别诱导OxLDL和乙酰化低密度脂蛋白(AcLDL)负载小鼠腹腔巨噬细胞所形成的巨噬泡沫细胞, 观察其胆固醇外流情况。(2)分离正常及apoE基因缺陷(E⁰)小鼠腹腔巨噬细胞, 以AcLDL负载形成巨噬泡沫细胞, 分别以LPC及apoAI作为诱导物, 观察其胆固醇外流情况。结果:(1)apoAI能引起AcLDL组巨噬泡沫细胞胆固醇大量外流, 而OxLDL组外流明显受阻。(2)LPC能引起正常组小鼠腹腔巨噬泡沫细胞胆固醇外流, 且呈剂量效应关系, 但E⁰组未见明显外流;apoAI能引起两组的胆固醇外流, 且外流量显著高于LPC。结论:(1)OxLDL能造成胆固醇外流途径受阻。(2)LPC能促进巨噬泡沫细胞胆固醇外流, 可能主要通过apoE途径来进行。     

LAG-3 (CD223) reduces macrophage and dendritic cell differentiation from monocyte precursors

Major histocompatibility complex (MHC) class II molecules expressed on monocytes may play a role in the control of differentiation of antigen-presenting cells. A soluble LAG-3 (CD223) molecule (sLAG-3) is a natural, high-affinity ligand for MHC class II. It is known to induce maturation of monocyte-derived dendritic cells in vitro and is used as a vaccine adjuvant to induce CD4 T helper type 1 responses and CD8 T-cell responses in vivo. Here, we demonstrate that sLAG-3 (but not an MHC class II-specific monoclonal antibody) reduces the differentiation of monocytes into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as their differentiation into dendritic cells in the presence of GM-CSF and interleukin-4, as shown by a decrease in CD14 and CD1a expression, respectively. Dendritic cells derived from monocytes in the presence of sLAG-3 showed impaired antigen-presentation function, as assessed by the reduced capability to induce proliferation of T cells. Our results suggest that activated LAG-3(+) lymphocytes present at sites of inflammation may reduce the differentiation of monocytes into macrophages or fully competent antigen-presenting dendritic cells, thus limiting the magnitude of the ongoing T-cell immune responses.     

Ghrelin inhibits foam cell formation via simultaneously down-regulating the expression of acyl-coenzyme A:cholesterol acyltransferase 1 and up-regulating adenosine triphosphate-binding cassette transporter A1

BackgroundGhrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), revealed cardioprotective effects in both experimental models and human. There is far less information on the mechanisms that produce antiatherogenic effects. We assessed the expression of acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) and  adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1), which have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation, in THP-1-derived foam cells in the presence of various concentration of ghrelin.MethodsAfter 48 h of culture in the presence of phorbol myristate acetate, THP-1 monocytes differentiated to macrophages. After another 24 h of culture with ox-LDL, the differentiated cells transformed to foam cells. Different concentrations of ghrelin and other intervention factors were added, respectively. The expression of ACAT-1 and ABCA1 was detected by a technique in molecular biology. The content of cellular cholesterol was measured by zymochemistry via a fluorospectrophotometer.ResultsGhrelin could down-regulate the expression of ACAT-1 and up-regulate the expression of ABCA1 in a dose-dependent manner simultaneously. Ghrelin also decreased cellular cholesterol content and increased cholesterol efflux. These effects could be abolished by the specific antagonist of GHS-R and a peroxisome proliferator-activated receptor γ (PPARγ)-specific inhibitor, respectively.ConclusionsThe results suggest that ghrelin inhibited foam cell formation via simultaneously down-regulating the expression of ACAT-1 and up-regulating ABCA1. Those effects may be achieved via pathways involving GHS-R and PPARγ.     

CpG-ODN对婴幼儿喘息性支气管炎外周血单个核细胞T-bet表达的影响

目的：探讨CpG-ODN干预对婴幼儿喘息性支气管炎外周血单个核细胞（PBMC）T-bet表达和IFN-γ分泌的影响。方法：对28例喘息性支气管炎（喘支组）患儿于急性期和恢复期分别抽取抗凝静脉血并分离PBMC,以终浓度为10μg/ml的B型CpG-ODN刺激48小时,分别应用RT-PCR和Western blot检测CpG-ODN刺激前后患儿PBMC的T-bet mRNA和蛋白表达情况,以ELISA检测PBMC培养上清IFN-γ的变化,同时设立26例健康婴儿对照组。结果：与正常对照组比较,喘支患儿PBMC表达T-bet mRNA及蛋白明显减少（P〈0.05）,应用CpG-ODN刺激后,两组小儿PBMC表达T-bet均显著上调,但产生IFN-γ无明显增多,对照序列ODN对T-bet表达及IFN-γ的分泌无明显影响。相关分析表明,喘支患儿T-bet mRNA与IFN-γ分泌呈正相关（r=0.57,P〈0.01）,CpG-ODN刺激后,二者相关性明显下降（r=0.19,P〉0.05）。结论：T-bet在喘支患儿PB-MC中表达减少,提示T-bet表达缺陷参与了喘支的免疫病理,B型CpG-ODN作为一种免疫调节剂,可有效上调T-bet的表达,但不能诱导IFN-γ的产生。     

Effects of macrophage colony-stimulating factor (M-CSF) on anti-fungal activity of mononuclear phagocytes against Trichosporon asahii

Trichosporon asahii is an emerging opportunistic pathogen in immunocompromised patients. Little is known about the mechanisms of host defence against T. asahii. We investigated the fungicidal activity of human peripheral blood monocytes and murine peritoneal macrophages against T. asahii isolates, and the effects of M-CSF on the anti-fungal activity of mononuclear phagocytes. We also established a neutropenic mouse model of disseminated trichosporonosis with T. asahii. M-CSF enhanced the phagocytic fungicidal activity of mononuclear cells, and infected mice treated with human M-CSF at 10 x 106 U/kg showed a significant improvement in survival rate, with fewer fungal colony counts in the lung compared with control mice. Mice treated with human M-CSF showed higher concentrations of tumour necrosis factor-alpha (TNF-alpha) in the lung and plasma compared with control mice. The survival rate was significantly reduced in mice treated with anti-mouse TNF-alpha. Our results showed that M-CSF enhanced the fungicidal activity of mononuclear phagocytes partly by production of TNF-alpha, and suggest that the administration of M-CSF to patients with disseminated trichosporonosis may be a useful adjunct to conventional anti-microbial therapy and prophylaxis.     

Ghrelin对THP-1细胞泡沫化过程中酰基辅酶A：胆固醇酰基转移酶1表达的影响

目的： 研究单核/巨噬细胞分化成为泡沫细胞过程中ghrelin对人酰基辅酶A:胆固醇酰基转移酶1(ACAT-1)表达以及细胞内胆固醇酯的影响。方法: 体外培养人源单核细胞系（THP-1）,由佛波酯（PMA）作用使其分化为巨噬细胞,后者可在氧化低密度脂蛋白（ox-LDL）存在条件下进一步转变为泡沫细胞。巨噬细胞加入不同浓度的ghrelin预孵2 h后再加入ox-LDL,油红O染色法观察细胞内脂滴含量,运用RT-PCR法检测ACAT-1 mRNA水平,Western blotting法检测ACAT-1蛋白表达,采用酶法,通过荧光分光光度计检测细胞内胆固醇酯含量。结果: Ghrelin可明显减少THP-1源性泡沫细胞内脂滴的形成,显著降低细胞内胆固醇酯含量,并能显著降低单核/巨噬细胞泡沫化过程中ACAT-1 mRNA 水平和蛋白表达,且此干预作用呈浓度依赖性。结论: Ghrelin可能通过ACAT-1转录翻译水平的下调延缓动脉粥样硬化的发生。     

氧化低密度脂蛋白诱导巨噬细胞泡沫化氧化损伤后血管紧张素转化酶2的表达变化及意义

目的观察氧化低密度脂蛋白(ox-LDL)诱导巨噬细胞泡沫化后血管紧张素转化酶2(ACE2)的表达变化与氧化应激损伤的关系,探讨ACE2的抗氧化作用。方法用不同浓度ox-LDL诱导小鼠巨噬细胞RAW 264.7泡沫化,油红O染色观察巨噬细胞内脂质堆积变化,并定量分析细胞内脂滴含量,通过检测细胞上清中丙二醛(MDA)含量鉴定细胞氧化损伤程度,实时定量聚合酶链反应(Real-time PCR)检测ACE2及MAS m RNA表达,化学比色法检测超氧化物歧化酶(SOD)活性与还原型谷胱甘肽(GSH)的含量。结果不同浓度(20、40、60、80mg/L)ox-LDL处理巨噬细胞24 h,油红O染色可见巨噬细胞变大、变圆,胞质内脂滴含量呈剂量依赖性增加;用60mg/L ox-LDL诱导巨噬细胞泡沫化模型组中MDA含量较对照组明显升高(P0.01);Real-time PCR结果显示,模型组ACE2和MAS m RNA表达水平较对照组显著减少(P0.05);与对照组比较,模型组中SOD活性与GSH含量显著降低(P0.05或P0.01)。结论巨噬细胞泡沫化氧化损伤进程与ACE2及其下游受体MAS表达下调密切相关,提示ACE2具有一定的抗氧化作用,其机制可能与SOD及GSH有关。     

PKC介导单核/巨噬细胞分化过程中间质标志物的表达变化

目的探讨单核/巨噬细胞分化过程中,PKC调控间质标志物表达变化的机制。方法以PMA诱导的单核细胞分化成巨噬细胞过程为研究对象,利用流式细胞术检测巨噬细胞表面标志物的表达;随后利用鬼笔环肽染色比较分化前后细胞长短轴的比例变化;利用Western blot法检测分化前后间质标志物相关蛋白的表达变化以及PKC抑制剂对间质标志物表达的影响。结果单核细胞向巨噬细胞分化过程中,间质标志物表达增加,并且这种变化受到PKC的调控。结论PKC介导单核/巨噬细胞分化过程中间质标志物的表达变化。     

Sida rhomboidea.Roxb aqueous extract down-regulates in vivo expression of vascular cell adhesion molecules in atherogenic rats and inhibits in vitro macrophage differentiation and foam cell formation

The present study evaluates efficacy of Sida rhomboidea.Roxb (SR) leaves extract in ameliorating experimental atherosclerosis using in vitro and in vivo experimental models. Atherogenic (ATH) diet fed rats recorded significant increment in the serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very LDL (VLDL), autoantibody against oxidized LDL (Ox-LDL), markers of LDL oxidation and decrement in high-density lipoprotein (HDL) along with increment in aortic TC and TG. The ex vivo LDL oxidation assay revealed an increased susceptibility of LDL isolated from ATH rats to undergo copper mediated oxidation. These set of changes were minimized by simultaneous co-supplementation of SR extract to ATH diet fed rats. Histopathology of aorta and immunolocalization studies recorded pronounced atheromatous plaque formation, vascular calcification, significant elastin derangements and higher expression of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and p-selectin in ATH rats. Whereas, ATH+SR rats depicted minimal evidence of atheromatous plaque formation, calcium deposition, distortion/defragmentation of elastin and accumulation of macrophages along with lowered expression of VCAM-1 and P-selectin compared to ATH rats. Further, monocyte to macrophage differentiation and in vitro foam cell formation were significantly attenuated in presence of SR extract. In conclusion, SR extract has the potency of controlling experimental atherosclerosis and can be used as promising herbal supplement in combating atherosclerosis.     

Sida rhomboidea.Roxb aqueous extract down-regulates in vivo expression of vascular cell adhesion molecules in atherogenic rats and inhibits in vitro macrophage differentiation and foam cell formation

The present study evaluates efficacy of Sida rhomboidea.Roxb (SR) leaves extract in ameliorating experimental atherosclerosis using in vitro and in vivo experimental models. Atherogenic (ATH) diet fed rats recorded significant increment in the serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), very LDL (VLDL), autoantibody against oxidized LDL (Ox-LDL), markers of LDL oxidation and decrement in high-density lipoprotein (HDL) along with increment in aortic TC and TG. The ex vivo LDL oxidation assay revealed an increased susceptibility of LDL isolated from ATH rats to undergo copper mediated oxidation. These set of changes were minimized by simultaneous co-supplementation of SR extract to ATH diet fed rats. Histopathology of aorta and immunolocalization studies recorded pronounced atheromatous plaque formation, vascular calcification, significant elastin derangements and higher expression of macrophage surface marker (F4/80), vascular cell adhesion molecule-1 (VCAM-1) and p-selectin in ATH rats. Whereas, ATH+SR rats depicted minimal evidence of atheromatous plaque formation, calcium deposition, distortion/defragmentation of elastin and accumulation of macrophages along with lowered expression of VCAM-1 and P-selectin compared to ATH rats. Further, monocyte to macrophage differentiation and in vitro foam cell formation were significantly attenuated in presence of SR extract. In conclusion, SR extract has the potency of controlling experimental atherosclerosis and can be used as promising herbal supplement in combating atherosclerosis.     

Fractalkine expression on human renal tubular epithelial cells: potential role in mononuclear cell adhesion

Fractalkine (CX3CL1) is a transmembrane molecule with a CX3C chemokine domain attached to an extracellular mucin stalk which can induce both adhesion and migration of leucocytes. Mononuclear cell infiltration at renal tubular sites and associated tubular epithelial cell damage are key events during acute renal inflammation following renal allograft transplantation. Using northern and Western blot analysis, we have demonstrated the expression of fractalkine message and protein by renal tubular epithelial cells in vitro. The expression was up-regulated by TNF-alpha, a key proinflammatory cytokine in acute rejection. Investigation of surface expression of fractalkine on cultured proximal tubular epithelial cells revealed only a subpopulation of positively staining cells. Immunohistochemistry revealed that only a proportion of tubules in renal allograft biopsies showed induction of fractalkine expression. Studies using a static model of adhesion demonstrated CX3CR1/fractalkine interactions accounted for 26% of monocytic THP-1 cell and 17% of peripheral blood natural killer cell adhesion to tubular epithelial cells, suggesting that fractalkine may have a functional role in leucocyte adhesion and retention, at selected tubular sites in acute renal inflammation. Thus, fractalkine blockade strategies could reduce mononuclear cell mediated tubular damage and improve graft survival following kidney transplantation.