VLDL和ox-LDL对单核巨噬细胞的作用及机制探讨

目的:探讨极低密度脂蛋白(VLDL)、氧化修饰的低密度脂蛋白(ox-LDL)对单核巨噬细胞的作用及机制。方法:分析VLDL、ox—LDL对单核巨噬细胞清道夫受体A(SRA)、VLDL受体(VLDLR)基因转录、细胞因子肿瘤坏死因子(TNF—α)、基质金属蛋白酶(MMP-9)蛋白质表达,以及细胞生长状态的影响。结果:①VLDL增加ox-LDL介导的巨噬细胞SRA mRNA及蛋白质表达,ox—LDL则能增加VLDL介导的巨噬细胞VLDLR基因转录;②VLDL与ox—LDL协同作用显著减少巨噬细胞抗脂质摄取载脂蛋白E分泌;③两种脂蛋白能诱导巨噬细胞TNF-α及MMP-9释放,并能抑制巨噬细胞凋亡,促进增殖。结论:VLDL、ox-LDL对单核巨噬细胞增生、细胞因子释放及脂蛋白受体表达均有协同作用,可能体内的情况多为两种脂蛋白的协同作用。     

过氧化体增殖物激活型受体-γ对巨噬细胞脂蛋白受体和炎症因子表达效应的研究

目的 探讨配体曲格列酮（Trog)活化的过氧化体增殖物激活型受体γ（PPAR-γ）对脂蛋白介导的巨噬细胞脂蛋白受体和炎症因子表达的影响。方法 采用密度梯度超速离心法获得低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL），用Cu^2 LDL氧化修饰为氧化LDL；通过免疫组织化学，Northern杂交和激光扫描共聚焦显微镜检测巨噬细胞清道夫受体A（SRA），VLDL受体（VLDLR），肿瘤坏死因子-α（TNF-α）和基质金属蛋白酶-9（MMP-9）以及PPAR-γ的表达或活化状态，结果 Trog活化的PPAR-γ能抑制巨噬细胞SRA，VLDLR，TNF-α和MMP-9的mRNA以及蛋白表达。结论 PPAR-γ抑制巨噬细胞脂质摄取和炎症因子表达的效应，对防治急性冠状动脉综合征可能起着一定的作用。     

PPARγ基因转录与单核巨噬细胞来源的泡沫细胞形成

目的 :探讨PPARγ基因转录在动脉粥样病变局部的作用。方法 :观察动脉粥样硬化局部PPARγ基因表达与巨噬细胞来源的泡沫细胞、清道夫受体A(SRA)分布的关系 ;分析PPARγ配体对氧化低密度脂蛋白 (ox LDL)介导的巨噬细胞SRA、PPARγmRNA表达、肿瘤坏死因子α(TNF α)和基质金属蛋白酶 9(MMP 9)释放的影响。结果 :PPARγ基因表达与巨噬细胞来源的泡沫细胞、清道夫受体A(SRA)的分布相似 ;PPARγ配体在增加巨噬细胞PPARγmRNA表达的同时 ,显著抑制了ox LDL介导的巨噬细胞SRAmRNA表达和TNF α、MMP 9的释放。结论 :PPARγ基因激活可抑制巨噬细胞SRAmRNA表达及细胞因子释放     

清道夫受体与糖尿病

清道夫受体 (SR)是主要存在于巨噬细胞膜表面的一种糖蛋白 ,能特异性结合并摄取氧化低密度脂蛋白等多种配体从而介导脂类代谢。本文主要介绍与糖尿病相关的SRA、SRB Ⅰ和CD36的结构、分布和功能 ,以及细胞因子等对其调节作用。在 2型糖尿病中CD36表达的水平增加 ,CD36和SRA Ⅰ在糖尿病动脉粥样硬化的形成中介导胆固醇的摄取 ,SRA Ⅱ作为糖基化终末产物的受体在糖尿病肾病中有促进肾小球硬化的作用。     

单核细胞株THP-1分化为泡沫细胞过程中清道夫受体A变化及阿托伐他汀的干预作用

目的 研究单核细胞株THP-1分化为巨噬细胞、泡沫细胞过程中细胞清道夫受体A（SRA）表达、单核细胞趋化蛋白-1（MCP-1）分泌及应用阿托伐他汀干预的情况。方法 佛波醇酯诱导THP-1细胞分化为巨噬细胞,将其分为对照组、OX-LDL组（泡沫细胞组）、OX-LDL＋阿托伐他汀组（再分为低、中、高浓度3组）。用ELISA法,测定细胞培养液中MCP-1浓度。将SRA特异性结合配体DiI-Ac-LDL与细胞孵育,应用荧光显微镜观察各组细胞SRA蛋白表达及活性情况。并将MCP-1浓度与SRA蛋白活性进行相关性分析。结果400倍光镜下观察到细胞株THP-1在佛波醇酯诱导下转变为泡沫细胞。与对照组相比,OX-LDL组MCP-1表达升高,6h后升高明显（P〈0．05）,12h达高峰（P〈0．01）,24h后逐渐下降（P〈0．01）。阿托伐他汀药物干预,呈剂量依赖性降低MCP．1水平。OX-LDL组SRA蛋白活性水平明显高于对照组（P〈0．01）。阿托伐他汀干预,呈剂量依赖性下调SRA蛋白活性水平。各组细胞12hMCP．1浓度与SRA蛋白活性水平呈明显正相关（r=0．683,P〈0．01）。结论SRA、炎症因子MCP-1在THP-1细胞分化为泡沫细胞过程中发挥重要作用,阿托伐他汀抑制MCP-1与SRA表达,可能是其抗动脉粥样硬化形成的重要机制。     

维拉帕米对人外周血单核巨噬细胞代谢低密度脂蛋白的影响

了解低密度脂蛋白的致动脉粥样硬化（ Ａ Ｓ）的机制及维拉帕米的影响。　方法：使用放射配基法观察人单核巨噬细胞对氧化低密度脂蛋白的代谢及维拉帕米的影响。　结果：氧化低密度脂蛋白在人单核巨噬细胞中的结合、摄取和降解较低密度脂蛋白增加。维拉帕米增加人单核巨噬细胞对氧化低密度脂蛋白和低密度脂蛋白的结合和摄取,不伴降解的增加。　结论：低密度脂蛋白经氧化后在人单核巨噬细胞中代谢发生改变,促进 Ａ Ｓ的形成,维拉帕米通过改变其代谢途径,具有抗 Ａ Ｓ的作用。     

氧化型低密度脂蛋白在人外周血单核巨噬细胞中的代谢

为探讨氧化型低密度脂蛋白在人外周血单核巨噬细胞中的代谢及其致动脉粥样硬化的机制，使用放射配基法观察了两种低密度脂蛋白在单核巨噬细胞中的代谢情况，并测定了细胞内胆固醇含量的变化。结果发现，人外周血单核巨噬细胞通过受体途径代谢低密度脂蛋白。该受体可饱和，最大结合量（Bmax）为274μg／g细胞蛋白，解离常数为35.7mg／L。低密度脂蛋白经氧化修饰后，人外周血单核巨噬细胞对其结合、摄取和降解均显著增加；与氧化型低密度脂蛋白共同孵育的人外周血单核巨噬细胞内总胆固醇含量明显高于低密度脂蛋白组。以上结果提示，低密度脂蛋白经氧化修饰后在人外周血单核巨噬细胞内的代谢途径发生改变并导致细胞内总胆固醇的堆积，促进了动脉粥样硬化的发生。     

高胰岛素状态下巨噬细胞PPARγ抗炎活性的变化

目的探讨高胰岛素状态下巨噬细胞过氧化物酶体增殖物激活受体γ（PPARγ）抗炎活性的变化。方法体外培养THP1细胞,诱导分化为巨噬细胞,采用吡格列酮和不同浓度胰岛素分组干预,ELISA法测定细胞培养上清液中IL6、TNFα、MMP9浓度,GelatinZymography法测MMP9活性。半定量RTPCR、Westernblot检测巨噬细胞PPARγ基因、蛋白表达。结果PPARγ配体吡格列酮（20μmol/L）作用24h可显著降低巨噬细胞IL6（P<0.01）、TNFα、MMP9的分泌（P<0.05）,抑制MMP9活性（P<0.05）。高胰岛素使吡格列酮抑制巨噬细胞分泌IL6、TNFα、MMP9的作用减弱,此作用与胰岛素浓度有关,呈浓度依赖性（0.25～0.5μmol/L）。而高胰岛素（0.1μmol/L）状态下巨噬细胞PPARγ基因、蛋白的表达均无显著变化。结论高胰岛素可减弱PPARγ配体抑制巨噬细胞分泌IL6、TNFα、MMP9的作用,但并未对巨噬细胞PPARγ基因、蛋白的表达产生影响,提示胰岛素对PPARγ的活性可能存在抑制。     

清道夫受体与糖尿病

清道夫受体(SR)是主要存在于巨噬细胞膜表面的一种糖蛋白，能特异性结合并摄取氧化低密度脂蛋白等多种配体从而介导脂类代谢。本文主要介绍与糖尿病相关的SRA、SRB-Ⅰ和CD36的结构、分布和功能，以及细胞因子等对其调节作用。在2型糖尿病中CD36表达的水平增加，CD36和SRA-Ⅰ在糖尿病动脉粥样硬化的形成中介导胆固醇的摄取，SRA-Ⅱ作为糖基化终末产物的受体在糖尿病肾病中有促进肾小球硬化的作用。     

PPAR-γ对单核/巨噬细胞酰基辅酶A:胆固醇酰基转移酶-1表达效应的研究

目的　研究配体罗格列酮活化的过氧化体增殖物激活型受体 γ(PPAR γ)对单核 /巨噬细胞转分化过程中酰基辅酶A :胆固醇酰基转移酶 1 (Acyl CoA :cholesterolacyltransferases,ACAT 1 )表达效应的影响。 方法　在RPMI1 640培养基中培养人单核细胞 (THP 1 ) ,加入佛波酯(PMA)培养 48h,细胞贴壁呈巨噬细胞样分化。运用免疫细胞化学、逆转录聚合酶链反应、蛋白质免疫印迹等方法 ,观察单核巨噬细胞转分化前后PPAR γ对ACAT 1mRNA和蛋白表达水平的影响。结果　单核巨噬细胞转分化前后ACAT 1表达增加 ,罗格列酮活化的PPAR γ可明显抑制A CAT 1的表达。结论　动脉粥样硬化事件的发生可能与ACAT 1表达增强有关。罗格列酮活化的PPAR γ可能通过抑制ACAT 1表达 ,巨噬细胞摄取脂质降低 ,从而减少泡沫细胞的形成 ,发挥其抗动脉粥样硬化的作用     

Absence of CD36 protects against atherosclerosis in ApoE knock-out mice with no additional protection provided by absence of scavenger receptor A I/II

AIMS: The role of scavenger receptors in atherogenesis is controversial as a result of conflicting reports and a recent hypothesis suggesting that scavenger receptor absence would enhance the pro-inflammatory, pro-atherogenic milieu. This study addresses the effect of combined absence of scavenger receptors CD36 and SRA I/II on atherosclerosis lesion development in the apolipoprotein E knock-out (apoE degrees ) model. METHODS: We created background-related strains of apoE degrees , scavenger receptor A I/II knock-out (SRA degrees )/apoE degrees , CD36 knock-out (CD36 degrees )/apoE degrees , and CD36 degrees /SRA degrees /apoE degrees mice that were >99% C57Bl/6. Four-week-old mice were fed a Western diet for 12 weeks and were assessed for lesion burden/morphology, risk factors for atherosclerosis, inflammatory mediators, and macrophage function. RESULTS: There was a 61 and 74% decrease in total aortic lesion area in CD36 degrees /apoE degrees males and females, respectively, compared with apoE degrees controls. The absence of SRA was protective (32% decrease in lesion) in female mice. The combined absence of CD36 and SRA provided no further protection in either gender. Macrophages from mice lacking CD36 had decreased pro-inflammatory characteristics and less migration to a pro-inflammatory stimulus. Plasma levels of cytokines/chemokines showed that CD36 degrees /apoE degrees and CD36 degrees /SRA degrees /apoE degrees mice had a less pro-inflammatory phenotype compared with apoE degrees and SRA degrees /apoE degrees mice. Oblivious mice in the apoE degrees background ruled out potential 'passenger gene' effects in the case of CD36. CONCLUSION: These results provide new insights into the pro-atherogenic mechanisms of CD36 by implicating processes other than modified lipoprotein uptake.     

Essential role for the (hepatic) LDL receptor in macrophage apolipoprotein E-induced reduction in serum cholesterol levels and atherosclerosis

Apolipoprotein E (apoE) is a high affinity ligand for several receptor systems in the liver, including the low-density lipoprotein (LDL) receptor, and non-LDL receptor sites, like the LDL receptor-related protein (LRP), the putative remnant receptor and/or proteoglycans. Although the liver is the major source of apoE synthesis, apoE is also produced by a wide variety of other cell types, including macrophages. In the present study, the role of the LDL receptor in the removal of lipoprotein remnants, enriched with macrophage-derived apoE from the circulation, was determined using the technique of bone marrow transplantation (BMT). Reconstitution of macrophage apoE production in apoE-deficient mice resulted in a serum apoE concentration of only 2% of the concentration in wild-type C57Bl/6 mice. This low level of apoE nevertheless reduced VLDL and LDL cholesterol 12-fold (P<0.001) and fourfold (P<0.001), respectively, thereby reducing serum cholesterol levels and the susceptibility to atherosclerosis. In contrast, reconstitution of macrophage apoE synthesis in mice lacking both apoE and the LDL receptor induced only a twofold (P<0.001) reduction in VLDL cholesterol and had no significant effect on atherosclerotic lesion development, although serum apoE levels were 93% of the concentration in normal C57Bl/6 mice. In conclusion, a functional (hepatic) LDL receptor is essential for the efficient removal of macrophage apoE-enriched lipoprotein remnants from the circulation and thus for normalization of serum cholesterol levels and protection against atherosclerotic lesion development in apoE-deficient mice.     

The LDL receptor is the major pathway for beta-VLDL uptake by mouse peritoneal macrophages

In order to determine the contribution of the low density lipoprotein receptor (LDL-R) to the removal of apoB-containing native lipoproteins by macrophages, we compared the uptake of beta-VLDL in peritoneal macrophages (MPM) from wild type mice and mice lacking the LDL-R. The d<1.006 g/ml lipoproteins obtained from apoE deficient mice fed a high fat diet were poorly degraded by macrophages and caused only a slight formation of CE in macrophages from both types of mice. On the other hand, d<1.006 g/ml lipoproteins obtained from LDL-R deficient mice fed a high fat diet, beta-VLDL with apoE, were avidly taken up by and markedly stimulated CE formation in wild type macrophages, but not in macrophages lacking the LDL-R. The degradation of 125I-labeled-apoE-containing beta-VLDL by wild type MPM was poorly inhibited by unlabeled human LDL, and beta-VLDL without apoE had no effects. In conclusion, we propose that the in vitro uptake of native apoE-enriched lipoproteins by murine macrophages is primarily mediated by the LDL receptor and not by other apoE-recognizing receptor systems such as: the LDL receptor related protein, the VLDL receptor or the triglyceride-rich lipoprotein receptor.     

Increased stability of phosphatase and tensin homolog by intermedin leading to scavenger receptor A inhibition of macrophages reduces atherosclerosis in apolipoprotein E-deficient mice

Intermedin, a novel member of calcitonin gene-related peptide family, is an endogenous cardiovascular-protective peptide. Because intermedin exists in human atherosclerotic plaque, we studied the role of intermedin in macrophage scavenger receptor A (SR-A)-mediated foam-cell formation and atherogenesis. In an in vitro foam-cell formation model (induced by acetylated low-density lipoprotein [AcLDL]) with mouse (C57BL/6J) macrophages, intermedin reduced AcLDL uptake and binding, decreased intracellular cholesterol content, and suppressed both mRNA and protein levels of SR-A. Simultaneously, intermedin increased phosphatase and tensin homolog (PTEN) protein levels by increasing PTEN phosphorylation and inhibiting ubiquitin-mediated PTEN degradation. These effects were blocked by the intermedin receptor antagonist or cAMP-protein kinase A inhibitors. PTEN overexpression mimicked the inhibitory effects of intermedin on SR-A expression and AcLDL uptake. However, knockdown of PTEN by short-hairpin RNA completely blocked all inhibitory effects of intermedin. Furthermore, in apolipoprotein E-deficient (apoE(-/-)) mice, 6-week intermedin infusion reduced AcLDL uptake and SR-A mRNA and protein levels and increased PTEN protein level in peritoneal macrophages. PTEN level was increased and SR-A expression decreased in parallel in macrophages in atherosclerotic lesions. Thus, intermedin inhibited atherosclerosis in apoE(-/-) mice. Increased stability of PTEN by intermedin leads to SR-A inhibition in macrophages, which ameliorates foam-cell formation and atherosclerosis in apoE(-/-) mice.     

辣椒来源外泌体样纳米囊泡通过ERK1/2通路拮抗巨噬细胞向泡沫细胞转化

目的]研究辣椒来源外泌体样纳米囊泡(CDELN)抑制氧化型低密度脂蛋白(ox-LDL)诱导THP-1源性巨噬细胞泡沫化的作用机制。 [方法]通过组织破碎、差速/超速离心及蔗糖密度梯度离心等步骤,分离提纯CDELN。利用ox-LDL刺激THP-1源性巨噬细胞24 h建立泡沫细胞模型,并进一步研究CDELN对巨噬细胞泡沫化的作用及其机制。采用激光共聚焦显微镜检测THP-1源性巨噬细胞对CDELN和人DiL标记乙酰化低密度脂蛋白(DiL-ac-LDL)的摄取;采用油红O染色检测细胞内胆固醇含量,通过分析细胞油红O染色阳性面积评估细胞内脂质累积影响;RT-qPCR和Western blot检测清道夫受体A(SRA)、脂肪酸转运蛋白(CD36)、凝集素样氧化型低密度脂蛋白受体1(LOX-1)、ATP结合盒转运体A1/G1(ABCA1/G1)以及丝裂原活化蛋白激酶(MAPK)通路p-ERK、p-p38 MAPK和p-c-Jun的mRNA和蛋白表达水平。 [结果]CDELN是大小均一、具有双层膜结构、富含蛋白质和核酸的外泌体样纳米囊泡,能被巨噬细胞摄取。DiL-ac-LDL摄取实验提示CDELN可抑制巨噬细胞的胆固醇摄取。油红O染色实验提示CDELN可减轻ox-LDL诱导下的胞内胆固醇蓄积。RT-qPCR及Western blot显示,CDELN能够显著降低ox-LDL诱导的THP-1源性巨噬细胞基质金属蛋白酶9(MMP-9)、SRA、CD36、LOX-1的mRNA水平,以及降低p-ERK、SRA、CD36、LOX-1蛋白水平。加入p-ERK激动剂Yoda1后,CDELN的保护作用被明显逆转。[结论]CDELN可显著抑制巨噬细胞泡沫化,该作用可能与抑制MAPK通路ERK1/2的磷酸化水平有关。     

Determinants of the uptake of very low density lipoprotein remnants by the perfused rat liver

The receptor-mediated uptake of very low density lipoprotein (VLDL) remnants by the rat liver was studied. Livers were perfused with native 125I-VLDL remnants, radiolabeled apo E-deficient remnants, and radiolabeled remnants that contained reductively methylated apo B and unmodified apo E. The specific uptake of the apo E-deficient remnants was 20% of that for the native remnants, whereas the specific uptake of the remnants containing unreactive apo B was 78% of the control value. This suggests that the apo E of VLDL remnants is the principal ligand for binding to the receptor, and in the absence of apo E, apo B may participate in binding. This conclusion is supported by the finding that dimyristoyl phosphatidylcholine (DMPC)- apo E complexes were effective in competing for the hepatic uptake of 125I-VLDL remnants. The intracellular distribution of radioactivity was analyzed by Percoll density gradient centrifugation. At five minutes after perfusion, radioactivity was associated with the plasma membrane and lysosomal fractions, and at 30 minutes most of the radioactivity was associated with the lysosomal fraction. Binding and internalization of VLDL remnants was also directly visualized by electron microscopy. Internalization proceeded by coated pit-coated vesicle formation with subsequent delivery to lysosomes. Our findings demonstrate that the apo E of VLDL remnants mediates binding to the hepatic receptor and that the internalization and degradation of VLDL remnants is by a similar pathway to that previously described for LDL.     

Recombinant human interleukin-3 and granulocyte-macrophage colony- stimulating factor show common biological effects and binding characteristics on human monocytes

Two human hemopoietic growth factors involved in monocytopoiesis, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied for their ability to stimulate blood monocytes and to bind to the monocyte membrane. Both cytokines maintained monocyte/macrophage numbers during long-term culture and increased cell size as compared with controls. Effects on cell numbers were present at low cytokine concentrations (6 to 20 pmol/L), whereas enhanced 3H-thymidine incorporation was observed only at higher concentrations (greater than or equal to 60 pmol/L). Autoradiographic studies showed only 1% to 3% of stimulated monocytes with nuclear grains. These results suggest that the primary mechanism for IL-3 and GM-CSF-induced maintenance of monocyte/macrophage numbers in humans is through an effect on cell survival. Surface receptors for both IL-3 and GM-CSF were studied by using 125I-labeled recombinant human (rh) cytokines and performing Scatchard analyses. Both cytokines showed curvilinear Scatchard plots, and computer analyses favored a two-site binding model. High-affinity binding data for 125I rhIL-3 (Kd 7.7 to 38.2 pmol/L; receptor number/cell 95 to 580) and for 125I rhGM-CSF (Kd 4.7 to 38.9 pmol/L; receptor number/cell 8 to 67) show similar binding affinities for the two cytokines but a lower receptor number/cell for 125I rhGM-CSF. Low-affinity binding characteristics for 125I rhIL-3 (Kd 513 to 939 pmol/L; receptor number/cell 179 to 5,274) and for 125I rhGM- CSF (Kd 576 to 1,120 pmol/L; receptor number/cell 130 to 657) show a similar pattern for the two cytokines. Specificity of 125I rhIL-3 and 125I rhGM-CSF binding to monocytes was established by the ability of the homologous cytokine to inhibit binding and the inability of a range of other cytokines to compete at 100-fold excess molar concentration. It is important, however, that binding of 125I rhIL-3 was partially inhibited by rhGM-CSF and that rhIL-3 partially inhibited binding of 125I rhGM-CSF to the monocyte membrane under conditions shown to prevent receptor internalization. The degree of inhibition varied between 25% and 80% in different experiments, and quantitative inhibition experiments showed that 1,000-fold excess concentrations of competitor failed to inhibit binding of the heterologous ligand completely. These results demonstrate that human IL-3 and GM-CSF have similar effects on growth and survival of human monocytes in vitro and suggest that these and other common biological effects may be mediated either through a common receptor or through distinct receptors associated on the monocyte membrane.