重组人钙调素对人脐静脉内皮细胞增殖作用的影响

本文探讨外源性重组人钙调素(recombinant human calmodulin,rhCaM)对人脐静脉内皮细胞(HUVEC)增殖作用的影响。采用基因重组技术在大肠杆菌DH5α中高效表达并纯化rhCaM。将HUVEC接种于96孔培养板,加入不同浓度rhCaM,用MTT比色法检测rhCaM对HUEVC细胞增殖作用的影响。结果表明rhCaM在0.04～0.4μg/ml浓度范围内可促进HUEVC的增殖,促增殖作用随细胞培养密度的增加而减弱,也与培养基中新生小牛血清的含量密切相关。故rhCaM对HUEVC增殖具有促进作用。     

链球菌组蛋白样蛋白对人脐静脉血管内皮细胞产生TNF-α的影响

研究链球菌HlpA对HUVEC产生TNF-α的作用。用1929成纤维细胞的细胞毒实验检测TNF-α活性。链球菌HlpA(0-80μg/ml)在体外以剂量和时间依赖方式促进HUVEC过量产生TNF-α(P<0.05)。LTA和HlpA的混合物对TNF-α的产生呈协同增强作用(P<0.05);抗HlpA和肝素则起抑制作用。     

剪切力对血管内皮细胞表达IL-8的影响

本文利用改进的流室装置, 通过精密蠕动泵提供剪切力, 选择5dyne/cm2、10dyne/cm2、15dyne/cm2三种剪切力, 施加于体外培养的血管内皮细胞, 作用不同时间, 用RIA的方法检测流体动力学对血管内皮细胞表达IL-8的影响.结果表示, 不同剪切力作用内皮细胞后, IL-8的表达量与剪切力大小及作用时间有关, 为时间依赖性增长, 在5dyne/cm2组和10dyne/cm2组中, IL-8表达量明显高于空白对照组, 而15dyne/cm2组与对照组相比有下降趋势, 提示高剪切力有可能抑制IL-8的分泌.通过观察不同大小剪切力对血管内皮细胞表达IL-8的影响, 为探讨剪切力对动脉粥样硬化的影响提供一些数据.     

Human Umbilical Vein Endothelial Cells Synthesize Functional C3, C5, C6, C8 and C9 In Vitro

Human endothelial cells (EC), cultured serum-free, synthesize de novo protein which increasingly bind to agarose beads (an alternative pathway activator), until a plateau phase is reached after 24-48 h. EC synthesize functional C3, C5, C6, C8 and C9, which were detected on co-cultured agarose beads, using relevant polyclonal anti-complement antibodies. Two monoclonal anti-C9 neoepitope antibodies (aE11, poly C9-MA) bound to the co-cultured beads, showing that the terminal complement complex (TCC) (C5b-9) was assembled on the beads. This also suggests that C7 is synthesized. There seems to be a positive correlation between the amount of agarose-bound labelled protein and agarose-bound complement. The results indicate that EC produce and secrete the components for the functional alternative and terminal pathways of complement.     

Effects of Citral on Lipopolysaccharide-Induced Inflammation in Human Umbilical Vein Endothelial Cells

Citral is an active compound of lemongrass oil which has been reported to have anti-inflammatory effects. In this study, we investigated the effects of citral on lipopolysaccharide (LPS)-induced inflammatory response in a rat model of peritonitis and human umbilical vein endothelial cells (HUVECs). LPS was intraperitoneally injected into rats to establish a peritonitis model. The HUVECs were treated with citral for 12 h before exposure to LPS. The levels of TNF-α and IL-8 were measured using ELISA. Western blotting was used to detect the expression of VCAM-1, ICAM-1, NF-κB, and PPAR-γ. The results showed that citral had a protective effect against LPS-induced peritonitis. Citral decreased the levels of WBCs and inflammatory cytokines TNF-α and IL-6. Citral also inhibited LPS-induced myeloperoxidase (MPO) activity in the peritoneal tissue. Treatment of HUVECs with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. LPS-induced VCAM-1 and ICAM-1 expression were also suppressed by citral. Meanwhile, we found that citral inhibited LPS-induced NF-κB activation in HUVECs. Furthermore, we found that citral activated PPAR-γ and the anti-inflammatory effects of citral can be reversed by PPAR-γ antagonist GW9662. In conclusion, citral inhibits LPS-induced inflammatory response via activating PPAR-γ which attenuates NF-κB activation and inflammatory mediator production.     

流体切应力作用强度对内皮细胞IL-8生成的影响

血管内皮细胞衬于血管腔的表面，是血流机械应力的主要感受者。切应力可以直接调节内皮细胞生物活性物质的合成和分泌，其中包括诱导内皮细胞生成IL-8，而且IL-8的生成量与切应力作用时间有关。为阐明内皮细胞IL-8的生成除了与切应力的作用时间有关外还与切应力的强度有关，我们用不同强度的流体切应力(2．09、4．61、6．19、8．51、10．50、12．59、14．41、17．22、18．32dyne／cm^2)处理培养的人脐静脉内皮细胞，然后采用双抗体夹心ABC-ELISA技术检测内皮细胞IL-8蛋白质的生成。结果显示：未用切应力处理的内皮细胞只有极少量的IL-8蛋白质生成；切应力处理内皮细胞后，低切应力(2．09dyne／cm^2)时IL-8蛋白质生成量明显增加，约为高切应力(18．32dyne／cm^2)时IL-8蛋白质生成量的6(作用5h)或7倍(作用6h)。IL-8蛋白质生成量与内皮细胞所施加的切应力强度呈反变关系；直线回归方程：5h时为y=760．12—36．06x，相关系数7=-O．978；6h时为y=781．87—36．66x，相关系数7=-O．980。式中：y为切应力作用下内皮细胞IL-8的生成量；x为施加于内皮细胞的切应力强度(dyne／cm^2)。不同的切应力作用时间(5h、6h)均表现出相同的IL-8蛋白质生成量随切应力强度的变化规律。提示流体切应力诱导内皮细胞生成IL-8的量，不仅与切应力的作用时间有关，而且IL-8的生成量与切应力强度有关。流体低切应力诱导内皮细胞IL-8的生成量急剧增高，可能在急性炎症和动脉粥样硬化的发生、发展过程中具有重要作用。     

流体切应力作用强度对内皮细胞IL一8生成的影响

血管内皮细胞衬于血管腔的表面,是血流机械应力的主要感受者.切应力可以直接调节内皮细胞生物活性物质的合成和分泌,其中包括诱导内皮细胞生成IL一8,而且IL一8的生成量与切应力作用时间有关.为阐明内皮细胞IL一8的生成除了与切应力的作用时间有关外还与切应力的强度有关,我们用不同强度的流体切应力(2.09、4.61、6.1 9、8.51、10.50、12.59、14.41、17.22、18.32 dyne/cm2)处理培养的人脐静脉内皮细胞,然后采用双抗体夹心ABC-ELISA技术检测内皮细胞IL一8蛋白质的生成.结果显示未用切应力处理的内皮细胞只有极少量的IL一8蛋白质生成;切应力处理内皮细胞后,低切应力(2.09dyne/cm2)时IL一8蛋白质生成量明显增加,约为高切应力(18.32 dyne/cm2)时IL-8蛋白质生成量的6(作用5 h)或7倍(作用6 h).IL一8蛋白质生成量与内皮细胞所施加的切应力强度呈反变关系;直线回归方程5 h时为y=760.12-36.06x,相关系数γ=-0.978;6 h时为y=781.87-36.66x,相关系数γ=-0.980.式中y为切应力作用下内皮细胞IL-8的生成量;x为施加于内皮细胞的切应力强度(dyne/cm2).不同的切应力作用时间(5 h、6 h)均表现出相同的IL-8蛋白质生成量随切应力强度的变化规律.提示流体切应力诱导内皮细胞生成IL一8的量,不仅与切应力的作用时间有关,而且IL-8的生成量与切应力强度有关.流体低切应力诱导内皮细胞IL-8的生成量急剧增高,可能在急性炎症和动脉粥样硬化的发生、发展过程中具有重要作用.     

Piper Sarmentosum Increases Nitric Oxide Production in Oxidative Stress: A Study on Human Umbilical Vein Endothelial Cells

OBJECTIVE:

Nitric oxide produced by endothelial nitric oxide synthase (eNOS) possesses multiple anti-atherosclerotic properties. Hence, enhanced expression of eNOS and increased Nitric oxide levels may protect against the development of atherosclerosis. Piper sarmentosum is a tropical plant with antioxidant and anti-inflammatory activities. This study aimed to investigate the effects of Piper sarmentosum on the eNOS and Nitric oxide pathway in cultured human umbilical vein endothelial cells (HUVECs).

METHODS:

HUVECs were divided into four groups: control, treatment with 180 μM hydrogen peroxide (H₂O₂), treatment with 150 μg/mL aqueous extract of Piper sarmentosum, and concomitant treatment with aqueous extract of PS and H₂O₂ for 24 hours. Subsequently, HUVECs were harvested and eNOS mRNA expression was determined using qPCR. The eNOS protein level was measured using ELISA, and the eNOS activity and Nitric oxide level were determined by the Griess reaction.

RESULTS:

Human umbilical vein endothelial cells treated with aqueous extract of Piper sarmentosum showed a marked induction of Nitric oxide. Treatment with PS also resulted in increased eNOS mRNA expression, eNOS protein level and eNOS activity in HUVECs.

CONCLUSION:

Aqueous extract of Piper sarmentosum may improve endothelial function by promoting NO production in HUVECs.     

Effects of Fumonisin B1 on Biomechanics and Cytoskeleton of Human Umbilical Vein Endothelial Cells

Objective Fumonisin B1(FB1)is an important mycotoxin in nature worldwide.The biomechanical properties of cells are closely related to their structure and function,and the cytoskeleton is the structural and functional basis of cells motility,and therefore,from a biomechanical point of view,the purpose of this study is to investigate the effects of FB1 on the biomechanical properties,migration capacity and cytoskeletal structure of human umbilical vein endothelial cells(HUVECs),which may lay an experimental foundation for further exploration of the toxicity mechanism of fumonisin.Methods HUVECs were cultured and treated with different concentrations of FB1.Then,CCK-8 kit was used to detect the effect of FB1 on the survival rate.The osmotic fragility of the cells was measured after treatment with different osmotic pressures for30 min.The cell membrane fluidity was measured by fluorescence polarization method.The cell electrophoretic mobility was measured by cell electrophoretic apparatus.The migration capacity of the cells was observed by scratch repair assay.The changes of reactive oxygen species and cytoskeletal structure were observed by confocal laser scanning microscopy.Finally,the mRNA and protein relative expression levels of cytoskeletal binding proteins were detected by real-time PCR,Western blotting and confocal laser scanning.Results The results of CCK-8 showed that FB1 could significantly inhibit the proliferation of HUVECs in a dose-and time-dependent manner.After treatment of HUVECs with FB1,the hypotonic resistance of the cell,cell surface charge,cell membrane fluidity and migration capacity were all weakened,while reactive oxygen species were significantly increased and the cytoskeletal structure was significantly reorganized.Furthermore,RTPCR results showed that the mRNA relative expression levels of cytoskeletal binding proteins,exception of actin,were down-regulated after treated with FB1.Besides,Western blotting and statistical analysis based on fluorescence intensity of laser confocal microscopy confirmed theses changes in protein level.Conclusions FB1 can significantly affect the biomechanical properties and motility of HUVECs,which may be directly correlated to the remodel of F-actin cytoskeleton,as well as the relative expression changes of cytoskeletal binding proteins.It is significant for further exploring the toxicity mechanism of fumonisin.     

生物材料对血管内皮细胞IL-8和TNF-α表达的影响

目的是研究不同生物材料对血管内皮细胞中细胞因子表达水平的影响，探讨与血栓形成有关的细胞因子mRNA表达变化与生物材料血液相容性之间的关联性。实验选择8％有机锡的聚氯乙烯为阳性对照，细胞培养用的聚苯乙烯为阴性对照，聚四氟乙烯、膨体聚四氟乙烯以及两种医用聚氨酯（PU-50和PU-60）作为实验材料，采用人脐静脉内皮细胞株ECV-304，通过细胞与不同材料的接触，运用逆转录聚合酶链反应（RT-PCR）方法，测定内皮细胞中与凝血功能密切相关的两种重要的细胞因子：白介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）的mRNA表达水平。结果显示：与阴性材料相接触的内皮细胞其IL-8和TNF-α的表达均呈阴性，与阳性材料接触的细胞因子表达均呈强阳性，两种聚氨酯材料尽管在细胞生长、附着和形态变化等方面经肉眼观察未见明显差异，但是两者在细胞因子的表达上却出现显著的不同，其中PU-60组表达水平明显高于PU-50组；聚四氟乙烯和膨体聚四氟乙烯材料表现出程度不等的表达增强，前者比后者更明显。将IL-8和TNF-α在上述6种材料中的表达情况进行秩相关分析，得到相关系数为r=0.88571（P〈0.05）。由此提示：血管内皮细胞对不同生物材料刺激所产生的反应在细胞水平和分子水平上可出现不完全一致的现象，后者显然较前者更为灵敏，通过检测血管内皮细胞中IL-8和TNF-α的mRNA表达改变，在一定程度上可推测生物材料血液相容性的优劣。     

Regulation of IL-8 production by complement-activated product,C5a,in vitro and in vivo during sepsis

Excessive complement-activated product complement 5a (C5a) has been implicated in the pathogenesis of sepsis development. Herein, we employed in vitro and in vivo models of sepsis to investigate the functional relationship between overtly produced C5a and IL-8. Our data revealed that C5a could strongly amplify IL-8 expression from human whole blood cells induced by LPS and other types of TLR agonists. ERK1/2 and p38, but not JNK, were mainly participated in signaling pathways for IL-8 production. In the whole blood stimulated by Escherichiacoli, C5a levels were quickly elevated and blockage of C5a significantly decreased E. coli-elicited IL-8 production. In the mouse model of sepsis induced by cecal ligation and puncture (CLP), the markedly increased keratinocyte-derived cytokine (KC) could be strongly suppressed by blockage of C5a. These data suggest that excessive C5a functions as a critical inflammatory mediator to enhance IL-8 production mainly through MAPK signaling pathways.     

Effect of Cholesterol Depletion on Interleukin-8 Production in Human Respiratory Epithelial Cells

Purpose

The lipid entities of cell membranes are components of the immune system and important mediators of inflammation. Despite increasing interest in the function of epithelial cells in inflammation, the role of cholesterol in this process has not been described. Here, we investigated the effect of cholesterol depletion on the inflammatory process in airway epithelial cells via the expression of interleukin (IL)-8 as a marker of inflammation.

Methods

A 549 cells were treated with 0.5% methyl-β-cyclodextrin as a selective cholesterol extractor. The IL-8 level was assessed by enzyme-linked immunosorbent assay and reassessed after cholesterol repletion. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the upstream signaling pathway for IL-8 production in cholesterol-depleted cells.

Results

We found a relationship between the amount of cholesterol in A 549 cells and inflammation of the airway. IL-8 production was increased in cholesterol-depleted A 549 cells and restored by cholesterol repletion. IL-8 production was decreased by pretreatment with the extracellular signal-regulated kinase (ERK) inhibitor U0126 but not with JNK inhibitor II or the p38 MAPK inhibitor SB202190.

Conclusions

Our findings suggest that inflammatory responses are increased in cholesterol-depleted epithelial cells via the MAPK signaling system, predominantly by the ERK pathway. We conclude that the lipid components of airwayepithelial cells may play a role in the inflammatory process.     

Modulation of Cytokine-Induced Production of IL-8 in Vitro by Interferons and Glucocorticosteroids

Interleukin-8 (IL-8) has been implicated in the pathogenesis of inflammation and cancer. Intracellular levels of cytokine-induced IL-8 in human umbilical vein endothelial cells (HUVEC) were modulated using interferons and steroids to further elucidate their mechanism. Basal and cytokine-induced production of IL-8 was studied using a novel ELISA application, flow cytometry, and RT-PCR. The intracellular amount of IL-8 increased after 6-h stimulation with TNF-alpha (30%) or IL-1beta (55%) which was doubled when Golgi transport was disrupted using monensin. IFN-gamma decreased the intracellular amount of IL-8 by 60% in both unstimulated and TNF-alpha-stimulated cells, but only when secretion was blocked using monensin. Dexamethasone inhibited the TNF-alpha-induced production by 33%, but had no effect in unstimulated cells. Our study indicated that both, dexamethasone and IFN inhibit TNF-alpha-induced upregulation of IL-8 at the mRNA level. It could be speculated that they inhibit IL-8 production by affecting different gene regulatory mechanisms.     

津力达颗粒预处理对高糖诱导小鼠胰岛微血管内皮细胞和人脐静脉内皮细胞增殖及凋亡的影响

目的:观察中药津力达颗粒预处理对高糖诱导的小鼠胰岛微血管内皮细胞(MS-1)及人脐静脉内皮细胞(HUVEC)增殖及凋亡的影响。方法:体外培养MS-1及HUVEC株,均随机分为正常浓度葡萄糖(5.5mmol/L)培养组(正常对照组)、高浓度葡萄糖(33mmol/L)培养组(高糖组)、不同浓度津力达颗粒(12.5μg/ml、25μg/ml、50μg/ml、100μg/ml、200μg/ml、400μg/ml、800μg/ml)预处理24h后再高糖培养组(津力达组),各组平行培养48h后收集细胞,MTT法检测细胞增殖水平,流式细胞术检测细胞凋亡,包括总凋亡指数(TAI)和早期凋亡指数(EAI)。结果:与正常对照组比较,高糖组MS-1及HUVEC增殖水平(OD值)下降(P0.01),TAI和EAI升高(P0.01)。与高糖组比较,津力达组MS-1及HUVEC增殖水平随药物浓度增加而升高(P0.01),至津力达浓度800μg/ml时与正常对照组差异无统计学意义(P0.05)。津力达组MS-1和HUVEC的TAI及EAI则随药物浓度增加而降低(P0.01),但尚不能回复至正常对照组水平。结论:中药津力达颗粒升高MS-1及HUVEC增殖水平,降低MS-1及HUVEC的TAI和EAI,从而可能对胰岛功能及血管内皮功能具有一定保护作用。     

Fibrinogen and Its Oxidized Form Induce Interleukin-2 Production in Cultured Endothelial Cells of Human Vessels

Oxidized fibrinogen was more potent than native fibrinogen in inducing interleukin-8 production in primary culture of human endothelial cells. The optimal concentration of oxidized fibrinogen was 3 mg/ml. The optimal time of UV irradiation was 17 min. Secretion of interleukin-8 was maximum during culturing of endothelial cells in a serum-free medium.     

流体切应力强度对内皮细胞IL-8基因表达的影响

内皮细胞位于血流与血管之间，内皮细胞调控的机械力相关反应已成为正常血管反应的一部分。切应力在调节内皮细胞功能上具有重要作用。流体切应力可以直接调节内皮细胞基因的表达，其中包括诱导内皮细胞表达IL－8，而且IL－8的表达量与切应力作用时间有关。 为阐明内皮细胞IL－8基因的表达除了与切应力的作用时间有关外还与切应力的强度有关，我们用不同强度的流体切应力（2．23、4．20、6．08、8．19、9．67、12．15、14．40、16．87、19．29dyne/cm^2）处理培养的人脐静脉内皮细胞，然后采用定量RT－PCR的方法检测内皮细胞IL－8 基因的表达情况。结果显示：未用切应力处理的内皮细胞没有IL－8基因的表达，切应力处理内皮细胞后，低切应力（2．23dyne/cm^2）时IL－8mRNA表达量明显增加为高切应力（19．29dyne/cm^2）时IL－8mRNA表达量的约68（作用1小h）或52倍（作用2h）。IL－8mRNA的表达量与内皮细胞所施加的切应务强度呈反变关系，直线回归方程，1h时为y=7.57-0.11x，相关系数r=7.97;2h时为y=7.92-0.10x，相关系数r＝0．96。式中：y为切应力作用下内皮细胞IL－8mRNA的表达量（拷贝数的对数值）；x为施加于内皮细胞的切应力强度（dyna/cm^2)。不同的切应力作用时间（1h,2h）均表现出相同的IL-8mRNA随切应力强度的变化规律。提示流体应力诱导内皮细胞表达IL－8，不仅与切应力的作用时间有关，而且IL－8的表达量与切应力强度有关。流体切应诱导内皮细胞IL－8mRNA的表达急剧增高，可能在炎症机制和动脉粥样硬化的发生、发展过程中具有重要作用。     

流体切应力作用时间对内皮细胞IL-8 基因表达的影响

内皮细胞对力学环境变化敏感，流体切应力可以直接调节内皮细胞基因的表达。为阐明内皮细胞白细胞介素-8（IL-8）基因的表达除受化学因子的调节外还受力学因素的影响，本文用流体切应力（2.23、4.20、6.08dyne/cm^2）处理培养的人脐静脉内皮细胞，然后采用定量RT-PCR的方法检测内皮细胞IL-8基因的表达情况。结果显示：未用切应力处理的内皮细胞没有IL-8基因的表达；切应力处理内皮细胞后，1h IL-8mRNA表达增加，2hIL-8mRNA表达量至最高值，3hIL-8mRNA表达量开始下降，4h后IL-8mRNA持续低表达；各实验组（2.23、4.20、6.08dyne/cm^2）均表现出相同的IL-8mRNA随时间的变化规律。提示流体切应力确可诱导内皮细胞表达IL-8，而且IL-8的表达量与切应力作用时间有关，呈双相性变化。流体切应力诱导内皮细胞表达IL-8，可能在急性炎症和动脉粥样硬化的发生、发展过程中具有重要作用。     

干扰素和肿瘤坏死因子对人脐静脉内皮细胞表达HLA-Ⅱ类抗原的影响

采用cell-ELISA法对IFN-α、γ及rTNFα单独或协同影响人脐静脉内皮细胞（HUVEC）表达HLA-Ⅱ类抗原作了定量观察。结果表明,IFNγ直接刺激 HUVEC可依浓度和时间依赖的方式提高其 HLA-Ⅱ类抗原表达量,而rTNFα、IFNα单独处理HUVEC无效。当rTNFα和IFNγ同时诱导时,对HUVEC表达HLA-DR、DP、DQ均表现抑制作用。但是,当先用IFNγ诱导HUVEC 24h后,再加入rTNFα则发现DR、DP、DQ的表达升高,起促进作用。     

Legumain对肿瘤细胞侵润力和血管内皮细胞管腔形成的作用

目的探讨肿瘤组织中过度表达的一种天冬酰胺肽链内切酶Legumain在肿瘤细胞侵润性生长和血管内皮导管形成中的作用及相关机制。方法采用体外细胞培养的方法,观察在缺氧条件下人乳腺癌细胞MDA MB231和人静脉血管内皮细胞HUVEC中Legumain的蛋白表达量：观察Legumain对MDA MB231细胞的侵润能力的影响以及人工合成的Legumain抑制物（asparaginly endopepidase inhibitor．AEPI）对人静脉血管内皮细胞HUVEC小管腔形成能力的影响;用免疫组织化学的方法观察Legumain与整合素（Integrin）β1的结合情况。结果Western—blot的结果表明。在缺氧条件下．体外培养的人乳腺癌细胞MDA MB231和人静脉血管内皮细胞HUVEC的Legumain蛋白表达量增加。应用免疫荧光双染色法对缺氧条件下培养的MDA MB231细胞进行染色．与正常条件下培养的细胞相比,缺氧条件下培养的MDA MB231细胞体积增大,胞浆内含有大量绿色的Legumain,成点状分布,并有部分Legumain分布在迁移中的细胞表面前缘,与红色的整合素β1重叠,呈现为黄色。在血管内皮细胞小管腔形成试验中,加入AEPI的实验组,HUVEC细胞的小管形成数量明显减少,分别为（13．2±0．85）,（5．33±0．35）,（0．57±0．23）,与对照组（21．3±1．73）相比差异均具有统计学意义（P〈0．001）。细胞的侵润能力试验结果表明,加入激活的Legumain后,实验组中的MDA MB231细胞的侵袭能力增高．分别为（170．90±59．27和（321．22±22．39）,与对照组（67．56±7．58）相比差异具有统计学意义（P〈0．05）。结论缺氧条件下．MDA MB231细胞株和HUVEC细胞株的Legumain表达增加,能促进肿瘤细胞的迁移和血管内皮细胞的小管形成。其分子机理之一可能是通过与整合素β1结合。