树突状细胞体外诱导抗肿瘤血管内皮细胞特异性免疫的研究

目的:探讨肿瘤内皮细胞抗原负载的树突状细胞(DC)诱导的细胞毒性T淋巴细胞(CTL)的特异性杀伤效应。方法:采用肿瘤细胞的培养上清诱导人脐静脉血管内皮细胞(HUVEC)增殖,制备肿瘤血管衍生的内皮细胞(TdEC)。用RTPCR检测肿瘤内皮标志物(TEM)的表达。制备TdEC的冻融抗原,负载从外周血中扩增的DC,用MTS比色法检测DC刺激自体淋巴细胞增殖的效应;用LDH法检测DC诱导的CTL的特异性杀伤效应。结果:TdEC可表达TEM1和TEM8。负载TdEC抗原的DC,可显著刺激自体淋巴细胞增殖。由其诱导的CTL对TdEC具有特异性的杀伤作用。在效靶比为20∶1和10∶1时,杀伤率分别为33%和27%,高于对照组的14%和10%。结论:TdEC抗原负载的DC,在体外可有效地诱导CTL产生,并特异性地杀伤TdEC。     

Ca2+与NO在过氧化氢所致正常成年大鼠肠系膜微血管通透性增高中的作用

 目的 观察内皮细胞Ca2+浓度及NO生成在过氧化氢所致正常成年大鼠肠系膜微血管通透性增高中的作用。方法 通过测定在体大鼠肠系膜微血管静水传导性观察微血管通透性变化。采用钙荧光指示剂(Fura 2-AM)、NO荧光指示剂(DAF-2 DA)标记在体微血管内皮细胞,并应用荧光显微镜检测细胞内钙或NO的荧光信号,观察H2O2作用下内皮细胞内钙离子浓度([Ca2+]i)、NO的变化。结果 H2O2可增加正常成年大鼠微血管通透性（正常对照的6.13±0.87倍,P<0.01）,同时增加微血管内皮细胞[Ca2+]i（714.58±144.70 nmol/L,P<0.01）,并促进内皮细胞NO的生成（正常对照荧光强度的1034.3%±44.3%,P<0.01）。Ca2+通道阻滞剂氯化镧可抑制H2O2所引起的微血管通透性增加（P<0.01）及内皮细胞[Ca2+]i升高（P<0.01）。NOS抑制剂AP-Cav-1可抑制H2O2所引起的微血管通透性增加（P<0.01）,但对H2O2的Ca2+增加作用无影响。结论 H2O2所致的通透性增加与细胞内Ca2+增加、NO的产生增多有关。     

Effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide concentration in normal and high-cholesterol fed rabbits

Introduction

Metabolic acidosis is present in end stage renal disease. There is a link between enhanced endothelial permeability and accelerated atherosclerosis. In this study, we investigated the effect of experimentally induced metabolic acidosis on aortic endothelial permeability and serum nitric oxide (NO) concentration in normal and high-cholesterol fed rabbits.

Material and methods

Twenty-four male rabbits were divided into four groups: normal, hypercholesterolemic, acidemic, and hypercholesterolemic plus acidemic. Acidosis and hypercholesterolemia were induced by drinking water containing ammonium chloride (NH₄Cl), and cholesterol-rich animal chow (1%), respectively. After 6 weeks, blood samples were taken and endothelial permeability was measured using the Evans blue dye injection method.

Results

Hypercholesterolemic animals had higher aortic endothelial permeability compared with normal groups (16.18 ±0.91 µg EB/g tissue vs. 12.89 ±0.66 µg EB/g tissue, p < 0.05). Acidosis significantly increased endothelial permeability in the normal group (17.10 ±0.56 µg/g tissue vs. 12.89 ± 0.66 µg/g tissue; p < 0.05) but did not further increase endothelial permeability in hypercholesterolemic animals (16.18 ±0.91 µg EB/g tissue vs. 17.29 ±0.46 µg EB/g tissue; p > 0.05). Serum total cholesterol, low density lipoprotein (LDL) and NO concentrations in hypercholesterolemic animals were significantly higher than the normal group and acidosis could not change them either in the normal or in the high-cholesterol diet group.

Conclusions

Alterations of serum lipids and NO are not the main mechanism for accelerated atherosclerosis during metabolic acidosis. Acidosis increases aortic endothelial permeability at least in a normal diet which may be a possible mechanism for progression of atherosclerosis processes in end-stage renal disease.     

Nitric oxide reduces tumor cell adhesion to isolated rat postcapillary venules

Adhesion of circulating tumor cells to microvascular endothelium plays an important role in tumor metastasis to distant organs. The purpose of this study was to determine whether nitric oxide (NO) would attenuate tumor cell adhesion (TCA) to naive or lipopolysaccharide (LPS)-treated postcapillary venules. A melanoma cell line, RPMI 1846, was shown to be much more adhesive to postcapillary venules isolated from rat mesentery than to corresponding precapillary arterioles. Although venules exposed to LPS for 4 h demonstrated an increased adhesivity for the melanoma cells, TCA to LPS-treated arterioles was not altered. Isolated venules exposed to DETA/NO (1 mm), an NO donor, for 30 min prior to tumor cell perfusion prevented the increment in adhesion induced by LPS and attenuated TCA to naive postcapillary venules. While L-arginine (100 m), an NO precursor, failed to decrease TCA to naive postcapillary venules, this treatment abolished LPS-stimulated TCA to postcapillary venules. The effect of l-arginine was reversed by administration of N -nitro-l-arginine methyl ester (l-NAME, 100 m), an NO synthase (NOS) inhibitor. These observations indicate that both exogenous and endogenous NO modulate TCA to postcapillary venules. To assess the role of NO-induced activation of cGMP in the reduction in TCA produced by DETA/NO, two additional series of experiments were conducted. In the first series, LY-83583 (10 m), a guanylyl cyclase inhibitor, was shown to completely reverse the effect of DETA/NO on TCA to both naive and LPS-activated postcapillary venules. On the other hand, administration of 8-bromoguanosine 3,5-cyclic monophosphate (8-B-cGMP) (1 mm), a cell permeant cGMP analog, mimicked the effect of DETA/NO and reduced TCA to LPS-stimulated postcapillary venules. These data suggest that (a) tumor cells are more likely to adhere to postcapillary venules than to corresponding precapillary arterioles, (b) LPS enhances TCA to postcapillary venules, (c) both exogenously applied (DETAINO) and endogenously generated (l-arginine) NO attenuate the enhanced adhesion induced by LPS, but only DETA/NO reduced TCA to naive postcapillary venules, and (d) the NO-induced reduction in TCA to LPS-activated postcapillary venules occurs by a cGMP-dependent mechanism.[/p]     

一氧化氮生物利用度失调与动脉粥样硬化

内皮功能障碍与动脉粥样硬化(AS)密切相关。氧化应激、脂质浸润、炎性反应因子表达及血管张力改变等涉及一氧化氮(NO)生物利用度(内源性NO的生成和利用)的降低,在内皮功能障碍中发挥重要作用。精氨酸酶活性增强、非对称性二甲基精氨酸和同型半胱氨酸增加均能使NO生物利用度下降,促进AS发生发展。糖尿病、肥胖、慢性肾病和吸烟等通过多种方式影响NO生物利用度,参与AS的发生。     

尿酸抑制人脐静脉内皮细胞合成一氧化氮

目的 研究尿酸(UA)对人脐静脉内皮细胞(HUVEC)表达内皮型一氧化氮合酶(eNOS)及分泌一氧化氮(NO)的影响.方法 不同浓度UA(0、0.5、1、1.5及2 mg/L)及50 mg/L ox-LDL(阳性对照)分别作用HUVEC 24、48及72 h,用real-time PCR法测定HUVEC eNOS mRNA;Western blot法检测细胞eNOS蛋白;酶法检测上清液NO的含量.结果 UA 0.5 mg/L组eNOS mRNA表达水平明显高于对照组(P＜0.05);随着UA浓度升高(1、1.5及2 mg/L组),及其作用时间延长,HUVEC eNOS mRNA及蛋白表达水平及上清液NO分泌最相比对照均明显下降(72 h N02-/N03-2 mg/L组与对照组分别为0.52±0.18与1.00±0.10,P＜0.05),且趋势与ox-LDL组相同.结论 0.5 mg/L以上浓度的UA呈浓度及时间依赖性抑制HUVEC eNOS表达及NO合成,提示高浓度的UA可能损伤血管内皮功能.     

溶血磷脂酰胆碱对内皮细胞一氧化氮的生成的影响

目的：探讨溶血磷脂酰胆碱（Lysophosphatidylcholine,Lyso-PC)对培养的人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs)一氧化氮（nitric oxide,NO)的生成的影响。方法：采用NO酶法测定内皮细胞NO生成的变化。结果：内皮细胞NO的生成对Lyso-PC具有时间和剂量依赖性,结论：Lyso-PC可能通过抑制内皮细胞NO的生成而导致动脉粥样硬化损伤的形成。     

Down-regulation of focal adhesion kinase, pp125FAK , in endothelial cell retraction during tumor cell invasion

Although endothelial cell retraction is required before tumor cell invasion, its molecular mechanism still remains obscure. We previously demonstrated that conditioned medium (CM) derived from a human pan-creatic cancer cell line, PSN-1, induced endothelial cell retraction and facilitated tumor cell invasion. To investigate the molecular change of events in the transduction of extracellular signals during endothelial cell retraction, we examined the effect of the CM derived from PSN-1 cells on the tyrosine phosphorylation in endothelial cells. Immunoblot analyses revealed that the PSN-1 CM decreased tyrosine phosphorylation of a 120-130 kD protein, and induced the concomitant down-regulation of focal adhesion kinase, pp125^FAK , dur-ing endothelial cell retraction in time- and dose-dependent fashions. These changes preceded endothelial cell retraction and were reversible after removal of the CM. Further quantitative densitometric analyses demon-strated that the extent of decrea se in tyrosine phosphorylated 120-130 kD protein during the endothelial cell retraction was likely to be proportional to that of the down-regulation of pp125^FAK . A tyrosine phosphory-lated 120-130 kD protein immunoprecipitated by anti-phosphotyrosine antibody immunoreacted with anti-pp125^FAK antibody. These results suggested that decreased amount of a tyrosine phosphorylated 120-130 kD protein probably due to the down-regulation of pp125^FAK might be associated with the signal transduction pathway in the endothelial cells during their retraction. Furthermore, these findings were also observed in the CM from another four human cancer cell lines, suggesting the down-regulation of pp125^FAK in endothelial cells during tumor cell invasion. © Rapid Science 1998     

一个肿瘤区新生血管内皮细胞的标志物

以肝癌细胞培养上清诱导增生的人脐静脉内皮细胞作为抗原，免疫Ｂａｌｂ／ｃ小鼠，获得１株单克隆抗体（ｍＡｂ）ＢＶＥ－１。该ｍＡｂ具有与肿瘤区增生血管内皮细胞结合而不与正常组织血管内皮细胞结合的特点，其相应抗原的相对分子质量（Ｍｒ）为１５００００，为一种含唾液酸的糖脂蛋白。ｍＡｂＢＶＥ－１与内皮细胞结合后可抑制其生长、移动和血管样结构形成。这一抗原可能是血管新生时表达的一种特异性标志物，其抗体可望成为靶向肿瘤区血管的药物载体。     

Alterations in the activity and expression of endothelial NO synthase in aged human endothelial cells

This study was to investigate factors underlying the age-related decrease in NO production in vascular endothelial cells. The age-related changes in NO production, the activity and expression level of eNOS, and eNOS binding proteins, were studied in HUVECs.NO production in HUVECs significantly decreased in an age-dependent manner. The potentiation of NO production by l-Arg was significantly suppressed by L-NIO (eNOS-specific inhibitor) in young HUVECs and was suppressed by 1400W (iNOS-specific inhibitor) in aged HUVECs. The aged HUVECs had lower eNOS protein levels than young cells. eNOS phosphorylation at Ser-1177 (active) decreased gradually from PDL 23 through 40, and eNOS phosphorylation at Thr-495 (inactive) increased in aged cells. Changes of intracellular eNOS binding proteins, such as caveolin-1, pAkt, and Hsp90, as well as interaction between eNOS and eNOS binding proteins, indicated decreasing enzyme activity in aged HUVECs.Aging might decrease the activity as well as expression level of eNOS in HUVECs. And the decrease in eNOS activity probably implicated to the alterations in the regulatory binding proteins. For further study, it needs to be confirmed that the age-related change in the intracellular distribution of eNOS and the relative contribution of eNOS and iNOS on vascular dysfunction in aged endothelial cells.     

人endostatin体外诱导血管内皮细胞凋亡的实验研究

目的 探讨人endostatin抑制血管内皮细胞增殖的作用机制。方法 在含重组人endostatin蛋白和10%小牛血清的DMEM培养基中，培养人脐静脉内皮细胞ECV304。72h后，采用透射电镜，流式细胞术细胞周期分析和细胞核DNA的1.5%琼脂糖凝胶电泳，检测重组人endostatin蛋白作用后，血管内皮细胞的凋亡。结果 透射电镜下可见实验组ECV304细胞核染色质浓缩，边集，核碎裂及胞浆浓缩等，呈典型的凋亡细胞的形态学表现，流式细胞术细胞周期分析显示，在G1期峰前存在1个凋亡峰（20.6%)，1.5%琼脂糖凝胶电泳显示，细胞核DNA呈梯状，对照组ECV304细胞表达正常。结论 重组人endostatin蛋白可诱导血管内皮细胞凋亡，是其抑制血管内皮细胞增殖的原因之一。     

Effect of pro-inflammatory stimuli on tumor cell-mediated induction of endothelial cell adhesion molecules in vitro

The object of our study was the question about the relevance of the tumor surrounding inflammatory cells with respect to the metastatic potential of the tumor cells. To imitate the role of inflammatory cells, three colon carcinoma (HT-29, HRT-18, and SW-620), one breast carcinoma (MCF-7), and one melanoma (ST-ML-12) cell lines were treated with pro-inflammatory stimuli, LPS, TNF-alpha, or IL-1beta. HUVEC monolayers were then stimulated by the collected supernatants (SN) of the tumor cells, following washing out of the applied stimuli. Analysis of CAM expression on HUVEC was performed using cell enzyme immunoassay. E-selectin, VCAM-1, and, in part, ICAM-1 were significantly up-regulated on HUVEC by exposure to SN of all LPS-stimulated tumor cells. This was especially the case for the colon carcinoma cell lines. A minimal increase of expression of VCAM-1 was observed after exposure to SN from TNF-alpha-stimulated HT-29 and MCF-7 cells. IL-1beta stimulation had no effect on endothelial CAM expression. These observations indicate that LPS could play a crucial role in tumor metastasis by inducing the release of soluble factors from different tumor cell lines capable of up-regulating CAM expression. This might be of special significance in colon carcinomas, where a large source of bacterial LPS is available in the intestinal lumen.     

血流切应力调控内皮型一氧化氮合酶的分子机制

血流切应力（flowshear stress，rss）是生理或病理条件下调节血管内皮细胞产生一氧化氮的最重要的刺激因素。FSS对内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）的调控包括基因转录的调节、转录后的调节和翻译后的调节。eNOS基因转录以及转录后mRNA的稳定性能被FSS诱导加强。FSS通过游离钙离子浓度、磷酸化、eNOS相关蛋白以及细胞内易位等途径调节eNOS的催化活性。此外，FSS还能调控eNOS催化反应的辅助因子。     

Protective role of endothelial nitric oxide synthase

Nitric oxide is a versatile molecule, with its actions ranging from haemodynamic regulation to anti-proliferative effects on vascular smooth muscle cells. Nitric oxide is produced by the nitric oxide synthases, endothelial NOS (eNOS), neural NOS (nNOS), and inducible NOS (iNOS). Constitutively expressed eNOS produces low concentrations of NO, which is necessary for a good endothelial function and integrity. Endothelial derived NO is often seen as a protective agent in a variety of diseases.This review will focus on the potential protective role of eNOS. We will discuss recent data derived from studies in eNOS knockout mice and other experimental models. Furthermore, the role of eNOS in human diseases is described and possible therapeutic intervention strategies will be discussed.     

Alcohol extracts of Echinacea inhibit production of nitric oxide and tumor necrosis factor-alpha by macrophages in vitro

It has been suggested that Echinacea has anti-inflammatory activity in vivo. Nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta are important mediators in the inflammatory response. The effect of alcohol extracts of E. angustifolia (EA), E. pallida (EPA) and E. purpurea (EP) on the production of these inflammatory mediators in both LPS-stimulated RAW 264.7 macrophages in vitro and murine peritoneal exudate cells (PECs) in vivo were investigated. As macrophages produce these inflammatory mediators in response to pathogenic infection, parallel cultures of macrophages were studied for phagocytosis and intracellular killing of Salmonella enterica. EPA and EP in vitro inhibited NO production and TNF-α release in a dose-dependent manner. RAW 264.7 cells treated with EA or EP showed decreased killing over 24 h, although EA enhanced bacterial phagocytosis. Upon bacterial infection, RAW 264.7 cells produce high levels of NO; however, an Echinacea-mediated decrease in NO production was observed. Echinacea alcohol extracts administered orally at 130 mg/kg per day for seven days had a weak effect on NO production and phagocytosis by LPS-stimulated PECs. The results indicated that all Echinacea species significantly decreased inflammatory mediators in vitro, however, only EA and EP reduced bacterial killing. Oral administration of Echinacea alcohol extracts did not adversely affect the development and anti-bacterial function of inflammatory PECs in vivo, however, NO production was decreased during bacterial infection of PECs.     

Nitric oxide attenuates vascular endothelial cadherin‐mediated vascular integrity in human chronic inflammation

In this study, we examined the role of nitric oxide (NO) in controlling vascular integrity mediated by vascular endothelial (VE)‐cadherin in chronic inflammation. Periapical granulomas were analysed for the expression of inducible NO synthase (iNOS) and VE‐cadherin, and more iNOS expression than VE‐cadherin was shown. Human umbilical vein endothelial cells (HUVECs) were stimulated with proinflammatory cytokines and lipopolysaccharide extracted from Porphyromonas gingivalis and it induced iNOS expression, whereas it reduced VE‐cadherin expression, compared with negative controls. On the other hand, pre‐incubation with 1400W, an iNOS‐specific inhibitor, markedly reduced iNOS expression in stimulated HUVECs and restored VE‐cadherin expression to its control level, suggesting that vascular integrity was modulated in conjunction with the reduction of NO. Immunocytochemistry confirmed the functional role of NO in cultured HUVEC monolayers with or without 1400W. These data are consistent with a hypothesis suggesting that NO could attenuate VE‐cadherin‐mediated vascular integrity in human chronic inflammation.     

人内皮型一氧化氮合酶cDNA在COS-7细胞中的表达

本研究从人脐静脉血管内皮细胞中提取总RNA，采用逆转录PCR(RT-PCR)方法，扩增出人内皮型一氧化氮合酶(heNOS)cDNA，总长度为3731bp。将其克隆入pUCm—T载体质粒，序列分析表明，克隆所得片段含有完整开放阅读框架，与GenBank中heNOScDNA序列同源性达99．93％，在此基础上发现有若干核酸多态性。将该基因片段亚克隆到真核表达载体pcDNA3．0上，用脂质体转染法将pcDNA3．0／heNOS转染到COS-7细胞株，RT—PCR、Western blot分别检测到外源性eNOS基因在mRNA水平和蛋白质水平的表达，分析表明所表达的heNOS蛋白质分子量为145ku；L-^14C-精氨酸掺入同位素法检测证实所表达的eNOS蛋白具有生物学活性，能将精氨酸氧化为瓜氨酸。     

Heterogeneous expression of cell adhesion molecules by endothelial cells in ARDS

ARDS (acute respiratory distress syndrome) can be associated with septic shock and multiple organ failure caused by an uncontrolled systemic inflammatory response to Gram-negative bacterial infection. While in animal models the key role of the endothelial adhesion molecules ICAM-1, E-selectin, and VCAM in ARDS has been extensively studied, there are scarcely any corresponding pathomorphological studies of human lung tissue. Hence, little is known about whether there is a comparable, or even heterogeneous, expression pattern of these molecules in the human pulmonary vasculature. This study was therefore undertaken to investigate the immunohistochemical expression of the constitutively expressed PECAM (CD31) and the inducible molecules ICAM-1, E-selectin, and VCAM in ARDS lungs from patients who had died in septic shock induced by Gram-negative bacteria. While in all specimens (ARDS and normal lungs) there was homogeneous strong expression of PECAM in all vessels, ICAM-1 was clearly up-regulated in ARDS lungs. E-selectin and VCAM were not expressed by endothelial cells (ECs) in normal lungs, but in ARDS lungs there was strong expression of both molecules in larger vessels, while in the capillaries there was only mosaic-like weak expression of a few ECs. This immunohistochemical investigation demonstrates the induction and up-regulation of adhesion molecules in human ARDS lungs, comparable to that described in animal models. There is also markedly heterogeneous expression of E-selectin and VCAM, indicating toporegional differences in the function of pulmonary ECs.