双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞膜脂流动性的影响

目的探讨分叉双歧杆菌的完整肽聚糖（WPG）对巨噬细胞膜脂流动性的影响。方法首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,再用细胞膜磷脂荧光探针标记细胞,最后采用激光共聚焦显微镜结合激光漂白后荧光恢复技术检测巨噬细胞的膜脂流动性。结果WPG刺激组反映小鼠腹腔巨噬细胞膜脂流动性的平均荧光恢复率明显高于对照组（P〈0.01）。结论分叉双歧杆菌的完整肽聚糖可提高巨噬细胞膜脂流动性。     

单核细胞的原代分离方法优化及其炎症激活模型的构建

为了得到高纯度高得率的单核细胞(monocyte)并分析其在血栓发生中的作用,本研究利用10位健康人的血样分离出单个核细胞(peripheral blood mononuclear cell,PBMC),并通过免疫磁珠分选来获得单核细胞,测定PBMC与单核细胞的得率。通过制备血浆中的高丰度蛋白β2GPI,并与其抗体构成抗原抗体复合物来刺激细胞培养,通过荧光定量PCR检测细胞培养基中组织因子TF和TNF-α的含量,构建单核细胞炎症激活模型。实验结果显示单核细胞的得率较高,纯度和活性较好,且β2GPI纯化效果好。荧光定量PCR结果显示,与对照组相比,添加β2GPI抗原抗体复合物的实验组TF与TNF-αmRNA表达量明显增多。本实验表明纯化的单核细胞被充分激活,在细胞水平上为研究机体炎症反应和血栓形成的分子机理提供了炎症模型。     

癌细胞膜流动性与蛋白激酶C相关性的初步探讨

细胞膜流动性是细胞的重要物理性质,与细胞功能密切相关。为深入认识细胞膜与细胞功能的关系,研究癌症机理及寻找预防治疗的方法提供新的视角和实验数据,采用荧光漂白后恢复技术检测皮肤癌A431细胞和正常HACAT细胞的膜流动性,同时测定蛋白激酶C的活性及mRNA的表达。通过激光扫描共聚焦显微镜的荧光恢复曲线计算得知A431细胞的荧光恢复率和扩散系数均低于HACAT细胞,即A431细胞膜流动性低于HACAT细胞;而蛋白激酶C活性、mRNA表达量却高于正常细胞。以上差异都有统计学意义（P〈0．05）。由此推断癌细胞膜流动性与蛋白激酶C相互作用、相互影响,彼此之间存在密切的关系。     

膜蛋白氨基酸组成通过改变膜流动性影响粟酒裂殖酵母和酿酒酵母融合株耐酒精能力

实验显示,一种氨基酸混合液（含异亮氨酸、甲硫氨酸和苯丙氨酸,添加浓度分别为1.0、0.5和2.0g/L）能显著提高自絮凝酵母——粟酒裂殖酵母和酿酒酵母融合株SPSC的耐酒精能力。实验将菌体分别培养于添加（试验组）和未添加（对照组）该氨基酸混合液的条件下,然后收集菌体进行酒精（20%,V/V）冲击试验（30℃,9h）,结果,试验组的菌体尚有一半以上的存活细胞,而对照组的菌体全部死亡。通过对试验组和对照组的菌体细胞膜蛋白质氨基酸组成分析发现,试验组的菌体耐酒精能力提高与所添加氨基酸组入菌体的细胞膜密切相关。以DPH为荧光探针的细胞膜流动性测定分析进一步揭示,氨基酸组入菌体的细胞膜后,细胞膜能有效抵抗高浓度酒精冲击诱发的膜流动性的提高,从而维持膜的稳定。因此,实验首次揭示膜蛋白氨基酸组成可通过改变膜流动性而影响酵母菌的耐酒精能力。     

ABC法在膜受体流动性测量中的应用

本文首次把ABC法应用于受体流动性测量中的膜表面受体荧光标记,利用FRAP(Fluorescence Recovery After Photobleaching)技术实现了细胞内吞过程中膜受体流动性变化的测量.实验用Con A—Biotin和Avidin—FITC(ABC法)标记巨噬细胞ConA受体,测量ConA刺激不同时间细胞膜表面受体的荧光强度、扩散系数和荧光恢复率的变化.结果显示ABC标记法适合于测量细胞内吞过程中膜表面受体的流动性变化,且具有较高的灵敏度高;巨噬细胞受ConA刺激后,膜表面ConA受体的扩散系数和荧光恢复率较静息状态时明显降低.     

豚鼠动脉粥样硬化模型形成机制:LDL-C代谢异常

目的建立豚鼠动脉粥样硬化模型并探讨其形成机制,同时与大鼠进行比较,阐明模型特点及优势。方法应用高脂饲料诱导方法,观察豚鼠和大鼠动脉粥样硬化病变的形成情况。HE染色法分析主动脉内膜-中膜厚度、内膜炎性细胞浸润和内膜表层斑块的形成情况;酶法检测血脂,酶联免疫吸附法测定血清Ox-LDL浓度,实时定量PCR检测肝脏LDL-R mRNA表达的变化,免疫组化检测血管内膜CD36蛋白表达的变化。结果与对照组比较,豚鼠模型组动脉内膜明显增厚,单核细胞、巨噬细胞浸润与聚集增加,大量的泡沫细胞聚集形成斑块,而大鼠模型组未出现类似动脉粥样硬化病理改变。机制研究表明豚鼠较大鼠易于诱发形成动脉粥样硬化原因主要在于豚鼠血清Ox-LDL水平明显升高,肝脏LDL-R mRNA表达下调,动脉内膜CD36蛋白表达明显增强等。结论与大鼠不同,经高脂饲料诱导10周后,豚鼠可形成典型动脉粥样硬化病变,其机制主要在于LDL-C代谢异常。     

肥胖动脉粥样硬化大鼠模型的建立及验证

目的:通过饮食及药物诱导营养性肥胖及动脉粥样硬化大鼠模型的建立及评价。方法:健康成年wistar大鼠随机分组,利用钙负荷联合高脂饮食的方法建立肥胖动脉粥样硬化大鼠模型;在模型建立不同时期分别检测各组实验大鼠身长、体重及腹围情况,并测量不同阶段大鼠皮下脂肪、腹腔内脏脂肪及胸主动脉血管旁脂肪组织质量;测量大鼠血清中甘油三酯、总胆固醇、低密度脂蛋白胆固醇及高密度脂蛋白胆固醇浓度变化;测量大鼠各部位脂肪含量变化;病理HE染色明确动脉粥样硬化形成状况。结果:模型各组大鼠血清总胆固醇及低密度脂蛋白胆固醇明显增高;病理HE染色证明模型组大鼠造模8周即可形成动脉硬化表现,并且伴随造模时间的延长,胸主动脉中膜厚度明显增加;模型组大鼠在造模16周开始体重、腹围、身体各部位脂肪组织含量较对照组明显增加,肥胖模型成功。结论:于研究16-24周时间成功构建了肥胖及动脉粥样硬化共病大鼠模型,为进一步研究肥胖及动脉粥样硬化的发生发展机制提供了可靠的动物模型。     

RFP134对UMR106细胞膜脂流动性的影响

癌细胞具有与正常细胞不同的膜脂流动性,导致细胞对生长因子和癌基因产物反应敏感,引起细胞增殖失控。本实验室从植物中发现一种二萜类活性物质－RFP134,在细胞周期和信号传递等多方面表现出有抑制癌细胞增殖,促进细胞分化的作用。本文以大鼠成骨肉瘤细胞（UMR106）和下鼠成骨细胞为模型,研究其对癌细胞膜脂流动性的影响。细胞系UMR106由美国麻省总医院内分泌室赠送。成骨细胞由本实验室分离培养。以不同浓度（20、40、60、80、100μM／L）的RFP134,在同一时间处理细胞,或以最适浓度（50μM／L）在不同时间作用于细胞。DH为荧光标记物,测得的荧光偏值和微粘度值为膜膜流动性指标。结果显示,无论在恒定的时间、以不同浓度的RFP134作用于UMR106细胞(Fig.1B）,或以恒定的浓度、在不同时间处理UMR106细胞（Fig.1D）,结果均表现为显著降低膜脂流动性。前者,RFP134作用于细胞时,细胞荧光偏振瑟微粘度值逐步升高,其变化呈量效关系;而后者,呈时效关系。但在最适浓度与最佳作用时间,荧光偏振值和微粘度值达饱和状态。在同样条件下,RFP134对正常成骨细胞的膜脂流动性影响小。即：荧光偏振值和微粘度值均在正常范围内保持恒定（Fig.1A;Fig.1C）RFP134降低癌细胞的膜脂流动性,从而改善了它的细胞膜功能,降低了它对生长因子的反应性,恢复了细胞对调节因子的正常反应。这可能是RFP134能够抑制癌细胞增殖,促进细胞分化的机制之一。     

巨噬细胞吞噬过程中膜磷脂流动性和受体流动性的关系

本文用ＦＲＡＰ（ｆｌｕｏｒｅｓｃｅｎｃｅｒｅｃｏｖｅｒｙａｆｔｅｒｐｈｏｔｏｂｌｅａｃｈｉｎｇ）技术,测量了静息状态和刀豆素Ａ刺激不同时间后巨噬细胞膜磷脂、ＣｏｎＡ受体扩散系数和荧光恢复率的变化。结果显示ＣｏｎＡ刺激后膜磷脂和ＣｏｎＡ受体的扩散系数和荧光恢复率均较静息状态的巨噬细胞明显降低,磷脂流动性的变化与ＣｏｎＡ受体流动性的变化呈正相关。提示受体介导内吞导致的膜磷脂流动性的降低,可能是由于配体与细胞膜上受体结合形成配体－受体复合体,增加了受体的负荷,使受体的流动性降低,进而使膜磷脂的流动性降低。巨噬细胞内吞过程中膜磷脂和ＣｏｎＡ受体流动性的降低,可能还与ＣｏｎＡ刺激后巨噬细胞胞浆ｐＨ值有关。     

针刺法作用兔血管再狭窄模型的病理学研究

目的:探讨针刺作用动脉再狭窄的病理学改变,为针刺临床治疗冠状动脉成形术后再狭窄提供理论依据.方法:应用PTCA球囊导管对实验兔行双侧髂动脉内膜剥脱建立动脉粥样硬化模型术后,狭窄者再行PTCA球囊扩张术以建立再狭窄模型,动物组织进行HE染色,观察针刺后髂动脉,冠状动脉及主动脉内膜的形态学变化.结果:针刺组主动脉、髂动脉及冠状动脉脂斑面积与模型组差异不显(P＞0.05);内膜增生程度均较轻,内膜中VSMC、泡沫细胞较少;动脉内膜及中膜面积与模型组差异明显(P＜0.01).结论:针刺法对动脉血管再狭窄具有一定的防治作用.     

Cellular cholesterol regulates monocyte deformation

The role of cholesterol content on monocyte biomechanics remains understudied despite the well-established link between cholesterol and monocytes/macrophages in atherosclerosis, and the effect on other cell types. In this work, we have investigated the effect of cholesterol on monocyte deformability and the underlying molecular mechanisms. We altered the baseline cholesterol in human monocytic cell line THP-1, and investigated the changes in monocyte deformability using a custom microfluidic platform and atomic force microscopy. We observed that the cholesterol depletion lowered deformability while enrichment increased deformability compared to untreated cells. As a consequence of altered deformability, cholesterol depleted cells spread more on collagen-coated surfaces with elongated morphology, whereas cholesterol enriched cells had a more rounded morphology. We observed that the decreased deformability in cholesterol depleted cells, despite an increase in the fluidity of the membrane, is due to an increase in phosphorylation of Protein Kinase C (PKC), which translates to a higher degree of actin polymerization. Together, our results highlight the importance of biophysical regulation of monocyte response to cholesterol levels.     

Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Lipopolysaccharide exposure during purification of human monocytes by adherence increases their recovery and cytolytic activity

Exposure of monocytes to lipopolysaccharide (LPS) during, but not after, adherence purification increased their cytolytic activity in short-term 51Cr-release assays against K562 target cells. In the absence of LPS only a minority of monocytes could be recovered by adherence. With 1 ng/ml to 10 micrograms/ml LPS present during the 1-hr adherence procedure, however, monocytes spread more extensively on serum-coated plastic and glass surfaces and virtually all of the monocytes in a mononuclear leukocyte preparation were recovered in the adherent fraction. While increasing the recovery of monocytes threefold, LPS exposure during adherence also increased monocyte purity as assessed by peroxidase staining, morphology, and indirect immunofluorescence with monoclonal Mo2. The proportion of Leu-11-positive NK cells in the adherent fraction did not change. Depletion of NK cells by treatment with anti-Leu-11b and complement eliminated cytolytic activity from the nonadherent, but not from the adherent, fraction isolated with LPS. Thus, addition of LPS during adherence produced a monocyte preparation with enhanced cytolytic activity not attributable to NK contaminants. To test whether LPS caused production of lymphokines that activate monocytes, we tested supernatants of unseparated mononuclear leukocytes for the capacity to stimulate purified monocytes for cytolysis. Such supernatants stimulated monocytes more effectively than LPS alone. We conclude that LPS stimulates monocytes for cytolysis most effectively during adherence purification because LPS allows the recovery of weakly adherent monocytes with high cytolytic capacity; also, LPS may stimulate production of lymphokines that further augment monocyte cytolytic activity.     

Lipid droplet: A functionally active organelle in monocyte to macrophage differentiation and its inflammatory properties

Lipid droplets (LDs) perform several important functions like inflammatory responses, membrane trafficking, acts as secondary messengers, etc. rather than simply working as an energy reservoir. LDs have been implicated as a controlling factor in the progression of atherosclerosis followed by foam cell formation that derives from macrophages during the differentiation process. However, the role of LDs in monocyte differentiation or its further immunological function is still an area that mandates in-depth investigation. We report that LD dynamics is important for differentiation of monocytes and is absolutely required for sustained and prolonged functional activity of differentiated macrophages. In THP-1 cell line model system, we elucidated that increase in total LD content in monocyte by external lipid supplements, can induce monocyte differentiation independent of classical stimuli, PMA. Differential expression of PLIN2 and ATGL during the event, together with abrogation of de novo lipogenesis further confirmed the fact. Besides, an increase in LD content by free fatty acid supplement was able to exert a synergistic effect with PMA on differentiation and phagocytic activity compared to when they are used alone. Additionally, we have shown Rab5a to play a vital role in LDs biosynthesis/maturation in monocytes and thereby directly affecting differentiation of monocytes into macrophages via AKT pathway. Thus our study reveals the multi-faceted function of LDs during the process of monocyte to macrophage differentiation and thereby helping to maintain the functional activity.     

Regulation of Monocyte Adhesion and Migration by Nox4

We showed that metabolic disorders promote thiol oxidative stress in monocytes, priming monocytes for accelerated chemokine-induced recruitment, and accumulation at sites of vascular injury and the progression of atherosclerosis. The aim of this study was to identify both the source of reactive oxygen species (ROS) responsible for thiol oxidation in primed and dysfunctional monocytes and the molecular mechanisms through which ROS accelerate the migration and recruitment of monocyte-derived macrophages. We found that Nox4, a recently identified NADPH oxidase in monocytes and macrophages, localized to focal adhesions and the actin cytoskeleton, and associated with phospho-FAK, paxillin, and actin, implicating Nox4 in the regulation of monocyte adhesion and migration. We also identified Nox4 as a new, metabolic stress-inducible source of ROS that controls actin S-glutathionylation and turnover in monocytes and macrophages, providing a novel mechanistic link between Nox4-derived H₂O₂ and monocyte adhesion and migration. Actin associated with Nox4 was S-glutathionylated, and Nox4 association with actin was enhanced in metabolically-stressed monocytes. Metabolic stress induced Nox4 and accelerated monocyte adhesion and chemotaxis in a Nox4-dependent mechanism. In conclusion, our data suggest that monocytic Nox4 is a central regulator of actin dynamics, and induction of Nox4 is the rate-limiting step in metabolic stress-induced monocyte priming and dysfunction associated with accelerated atherosclerosis and the progression of atherosclerotic plaques.     

The role of the blood monocytes in atherogenesis]

The data on morphofunctional features of monocytes in healthy persons and the role of mononuclear phagocytes in immunological and non-immunological mechanisms of atherogenesis were presented. The review contains an information on the influence of various humoral and cellular factors on blood monocyte interaction with arterial intima and on the possible reasons of disturbances of monocyte lipid clearance from vascular wall at atherosclerosis.     

Chlamydia pneumoniae-infected monocytes exhibit increased adherence to human aortic endothelial cells

Interactions between monocytes and endothelial cells play an important role in the pathogenesis of atherosclerosis, and monocyte adhesion to arterial endothelium is one of the earliest events in atherogenesis. Work presented in this study examined human monocyte adherence to primary human aortic endothelial cells following monocyte infection with Chlamydia pneumoniae, an intracellular pathogen associated with atherosclerosis by a variety of sero-epidemiological, pathological and functional studies. Infected monocytes exhibited enhanced adhesion to aortic endothelial cells in a time- and dose-dependent manner. Pre-treatment of C. pneumoniae with heat did not effect the organism's capacity to enhance monocyte adhesion, suggesting that heat-stable chlamydial antigens such as chlamydial lipopolysaccharide (cLPS) mediated monocyte adherence. Indeed, treatment of monocytes with cLPS was sufficient to increase monocyte adherence to endothelial cells, and increased adherence of infected or cLPS-treated monocytes could be inhibited by the LPS antagonist lipid X. Moreover, C. pneumoniae-induced adherence could be inhibited by incubating monocytes with a mAb specific to the human beta 2-integrin chain, suggesting that enhanced adherence resulted from increased expression of these adhesion molecules. These data show that C. pneumoniae can enhance the capacity of monocytes to adhere to primary human aortic endothelial cells. The enhanced adherence exhibited by infected monocytes may increase monocyte residence time in vascular sites with reduced wall shear stress and promote entry of infected cells into lesion-prone locations.     

Activation of ganglioside GM3 biosynthesis in human blood mononuclear cells in atherosclerosis

Using blood monocytes and lymphocytes from atherosclerotic patients and healthy subjects we have investigated a role of ganglioside GM3 in monocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC). The results showed that activity of GM3 synthase and cellular levels of ganglioside GM3 in blood mononuclear cells from atherosclerotic patients were several-fold higher than those from healthy subjects. In monocytes the activity of GM3 synthase was one order of magnitude higher than in lymphocytes from both groups studied; this suggests the major contribution of monocytes to enhanced biosynthesis and levels of GM3 in mononuclear cells in atherosclerosis. Enrichment of monocytes from healthy subjects with ganglioside GM3 by their incubation in the medium containing this ganglioside increased adhesion of these monocytes to HUVEC up to the level typical for monocytes from atherosclerotic patients. In addition, an increase in CD11b integrin expression comparable to that seen in lipopolysaccharide-activated monocytes was observed. It is suggested that in atherosclerosis the enhanced cellular levels of GM3 in monocytes and lymphocytes may be an important element of cell activation that facilitates their adhesion to endothelial cells and penetration into intima.     

Role of redox factor-1 in hyperhomocysteinemia-accelerated atherosclerosis

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. We have previously shown that homocysteine can induce monocyte chemoattractant protein-1 (MCP-1) secretion via reactive oxygen species (ROS) in human monocytes in vitro. In the present study, we investigated whether redox factor-1 (Ref-1) is involved in HHcy-accelerated atherosclerosis. We used a mild HHcy animal model, aortic roots and peritoneal macrophages were isolated for immunohistochemistry and Western blotting, from apoE-/- and C57BL/6J mice fed a high Hcy diet (1.8 g/L) for 4 or 12 weeks. Four-week HHcy apoE-/- mice showed more plaques and significantly increased immunostaining of Ref-1 and MCP-1 in foam cells, and HHcy mice showed enhanced Ref-1 expression in peritoneal macrophages. To explore the mediating mechanism, incubation with Hcy (100 microM) increased Ref-1 protein level and translocation in human monocytes in vitro. In addition, Hcy-induced NADPH oxidase activity mediated the upregulation of Ref-1. Furthermore, overexpressed Ref-1 upregulated NF-kappaB and MCP-1 promoter activity, and antisense Ref-1 reduced Hcy-induced NF-kappaB DNA-binding activity and MCP-1 secretion. These data indicate that Hcy-induced ROS upregulate the expression and translocation of Ref-1 via NADPH oxidase, and then Ref-1 increases NF-kappaB activity and MCP-1 secretion in human monocytes/macrophages, which may accelerate the development of atherosclerosis.