The role of oxidant-mediated pathways in the cytotoxicity of endothelial cells exposed to mesenteric lymph from rats subjected to trauma-hemorrhagic shock

Because gut-derived factors carried in mesenteric lymph are implicated in multiple organ dysfunction syndrome and have been shown to injure endothelial cells, we investigated several cellular pathways by which this process could occur. To accomplish this, mesenteric lymph (5%, v/v) collected at 1 to 3 h postshock from male rats undergoing trauma (5-cm laparotomy) and hemorrhagic shock (90 min of mean arterial pressure [MAP] of 30 mmHg; T/HS) was tested for endothelial cell cytotoxicity on human umbilical vein endothelial cells (HUVECs). Over 30 pharmacologic agents that had been reported to inhibit endothelial cell death were tested for their ability to prevent T/HS lymph-induced HUVEC cell death. These included agents documented to protect against oxidant-mediated, calcium-mediated, and arachidonic acid pathway-mediated endothelial cell injury and death. These pharmacologic inhibitors were preincubated with HUVECs for 1 h or were added to the HUVECs simultaneously with lymph, and were then incubated for 18 h. Controls were lymph alone, inhibitor alone, or medium alone. Mitochondrial tetrazolium (MTT) and LDH release assays were used to determine cell viability. The inhibitors that significantly protected HUVECs from the cytotoxicity of T/HS lymph (P < 0.001) included the antioxidant combination of vitamins C and E and the antioxidant-lipooxygenase inhibitor nordihydroguaretic acid (NDGA). These agents were equally effective when added simultaneously with lymph or preincubated with the HUVECs, suggesting an extracellular or membrane-bound process. In summary, the inhibitors that provided protection from toxic lymph appear to work at the membrane and are involved in limiting membrane peroxidation. Based on this study, it appears that an oxidant pathway is involved in T/HS lymph-induced endothelial cell injury and death.     

血管抑素对人脐静脉内皮细胞及体外微血管模型的作用

目的:观察血管抑素对人脐静脉内皮细胞及体外微血管模型的抑制作用。方法:实验于2005-01/2006-12在中国医科大学基础医学院实验病理研究室(省部级)完成。①采用改进的Jaffe式培养法体外培养人脐静脉内皮细胞。②MTT法检测不同质量浓度(1,2,4,8,16,32mg/L)、不同时间(24,48,72h)血管抑素对人脐静脉内皮细胞增殖的影响。③流式细胞仪检测不同质量浓度(2,4,8,16,32mg/L)的血管抑素对人脐静脉内皮细胞生长周期的影响。④建立肿瘤微血管体外生成模型,培养4,8,12h倒置显微镜观察血管网形成情况;血管网未形成之前加16mg/L血管抑素观察对血管形成的影响;肿瘤微血管体外生成模型经24h培养形成血管网后加16mg/L血管抑素观察对新生血管的影响。结果:①血管抑素对人脐静脉内皮细胞增殖的影响:8,16,32mg/L血管抑素能明显抑制人脐静脉内皮细胞的生长(P<0.01),血管抑素作用于人脐静脉内皮细胞24,48,72h后细胞明显受抑制(P<0.01),这种作用具有剂量-时间依赖性。②血管抑素对人脐静脉内皮细胞生长周期的影响:血管抑素作用于人脐静脉内皮细胞后能诱导细胞凋亡。③血管抑素对体外血管模型的影响:光镜下血管抑素可抑制新生血管的形成,且能破坏新生的血管网。结论:血管抑素可能抑制人脐静脉内皮细胞增殖,从而破坏新生血管形成,抑制肿瘤的生长和转移。     

Adherence of human basophils to cultured umbilical vein endothelial cells

The mechanism by which circulating human basophils adhere to vascular endothelium and migrate to sites of allergic reactions is unknown. Agents have been identified which stimulate the adherence of purified basophils to cultured human umbilical vein vascular endothelial cells (HuVEC). Treatment of HuVEC with interleukin 1, tumor necrosis factor (TNF), bacterial endotoxin, and 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in time and dose-dependent increases of adhesiveness for basophils. Coincubation of basophils and HuVEC for 10 min with C5a, formyl-methionyl-leucyl-phenylalanine, the calcium ionophore A23187, platelet-activating factor, TNF, and TPA also resulted in significant dose-dependent increases in basophil adherence; this effect resulted from activation of the basophil. Adherence of basophils to HuVEC was time and temperature dependent, required divalent cations, and was unaffected by glucocorticoids. Monoclonal antibody 60.3, directed against the beta-subunit of the leukocyte adherence complex CD18, inhibited the binding of basophils to HuVEC. Adherence of basophils to vascular endothelium may be important in initiating basophil infiltrates in vivo.     

Trauma/hemorrhagic shock mesenteric lymph upregulates adhesion molecule expression and IL-6 production in human umbilical vein endothelial cells

Trauma/hemorrhagic shock (T/HS) is associated with significant lung injury, which is mainly due to an inflammatory process, resulting from the local activation and subsequent interaction of endothelial cells and leukocytes. Adhesion molecules expressed by both cell types play a crucial role in the process of neutrophil-mediated endothelial cell injury. We have previously shown that mesenteric lymph duct ligation prevents T/HS-induced lung leukocyte infiltration and endothelial injury, suggesting that inflammatory factors originating from the gut and carried in the lymph are responsible for the lung injury observed following T/HS. Based on these observations, we hypothesized that inflammatory substances in T/HS lymph trigger lung injury by a mechanism involving the upregulation of adhesion molecules. To test this hypothesis, we examined whether T/HS mesenteric lymph induces the expression of E-selectin, P-selectin, and intracellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs). Furthermore, because the cytokine IL-6 is an important component of the endothelial inflammatory process, we investigated how T/HS lymph affects the production of IL-6 by HUVECs. Mesenteric lymph from T/HS rats increased both E- and P-selectin, as well as ICAM-1 expression on HUVECS, as compared to trauma/sham shock (T/SS) lymph or medium only groups. However, T/HS lymph failed to induce the shedding of E-selectin. In HUVECs treated with T/HS lymph, IL-6 concentrations were higher than HUVECs treated with T/SS lymph. These findings suggest that mesenteric lymph produced after hemorrhagic shock potentiates lung injury by the upregulation of endothelial cell adhesion molecule expression and IL-6 production.     

人脐静脉内皮细胞分离培养及鉴定技术

目的探讨人脐静脉内皮细胞(HUVEC)体外分离原代培养的方法,并总结对培养的细胞鉴定方法。方法通过胰酶灌注法从人脐带获取内皮细胞进行分离培养,并采用免疫组化法及光镜和透射电镜观察超微结构鉴定所获得的细胞系。结果分离的HUVEC在体外7-10天左右可长成单层,光镜下胞体为单层铺路石状排列。第VⅢ因子相关抗原的检测为阳性。透射电镜下观察培养的内皮细胞胞浆内可见Weibel-Palade小体。结论用胰酶灌注法是获得脐静脉内皮细胞的有效方法。本方法为血管内皮细胞的研究提供了实验模型。     

Toxicity of sitosterol to human umbilical vein endothelial cells in vitro.

The pathogenesis of the early development of atherosclerosis in sitosterolaemia is unknown. The effect of sitosterol on vascular endothelial cells in vitro was investigated by culturing human umbilical vein endothelial cells in the presence of up to 0.7 mmol l-1 of sitosterol. Liposomes were used to supply the high sterol concentrations. Exposure to 0.7 mmol l-1 of sitosterol for 72 h caused contraction of the endothelial cells and increased release of intracellular lactate dehydrogenase. After 96 h incubation the cells were partly detached from the substrate. At this time-point 0.35 mmol l-1 of sitosterol also caused perturbation of the endothelial cells. However, we could not confirm previous reports that tissue plasminogen activator production was enhanced by sitosterol.     

Serum from healthy pregnant women reduces oxidative stress in human umbilical vein endothelial cells

This study was conducted to compare the effects of serum from healthy pregnant women and that from pregnant women with pre-eclampsia on oxidative stress in endothelial cells in culture. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from 18 pre-eclamptic, 18 healthy pregnant and 18 healthy non-pregnant women for 24 h. The levels of reduced glutathione (GSH) and lipid peroxides (LPOs) were measured in endothelial cell lysates. Measurement of malondialdehyde in combination with 4-hydroxyalkenals has been used as an indicator of LPOs. Serum from healthy pregnant women decreased significantly the LPO content in HUVECs in comparison with serum from pre-eclamptic women and healthy non-pregnant women (30.7+/-6.6 compared with 39.3+/-10.9 and 41.0+/-12.7 pmol/mg of protein respectively; P<0.003 and P<0.01 respectively). No differences in GSH content between the three groups (18.3+/-2.1 nmol/mg of protein for healthy pregnant, 19.2+/-3.3 nmol/mg for pre-eclamptic and 18.3+/-2.0 nmol/mg for healthy non-pregnant women) were found. Thus serum from normal pregnant women contains a factor(s) that decreases oxidative stress in human endothelial cells. This mechanism might be altered in pre-eclampsia.     

休克淋巴液对大鼠肺微血管内皮细胞炎症介质表达的影响

目的 观察休克淋巴液对大鼠肺微血管内皮细胞(PMVECs)自由基及一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)表达的影响,进一步探讨休克淋巴液损伤PMVECs的机制.方法 原代培养大鼠PMVECs至第3代进行研究.无菌条件下复制大鼠重症失血性休克模型(动脉压40 mm Hg维持90 min,1 mm Hg=0.133 kPa).引流正常大鼠和休克大鼠肠系膜淋巴液及门静脉血,与PMVECs孵育6h,同时以胎牛血清(FBS)和无血清的DMEM培养液作为对照.逆转录-聚合酶链反应(RT-PCR)检测诱生型一氧化氮合酶(iNOS)、TNF-α和IL-6的mRNA表达;检测培养上清液中丙二醛(MDA)、NO、TNF-α和IL-6的含量.结果 体积分数为4%终浓度的休克淋巴液作用6 h后,PMVECs中iNOS、TNF-α和IL-6的mRNA表达以及培养上清液中MDA、NO、TNF-α和IL-6水平均显著高于空白对照组、正常淋巴液组、休克血浆组、正常血浆组和无血清对照组;且休克血浆作用PMVEC 6 h后的iNOS、TNF-α和IL-6的mRNA表达及培养上清液中NO水平均显著高于空白对照组、正常淋巴液组、正常血浆组和无血清对照组(P<0.05或P<0.01).结论 4%终浓度的休克淋巴液可致大鼠PMVECs中iNOS、TNF-α和IL-6的mRNA表达增强,促进自由基释放,从而诱导细胞损伤.     

油茶皂苷C对脐静脉内皮细胞缺氧及复氧损伤时的影响

目的:研究脐静脉内皮细胞(HUVEC)缺氧/复氧损伤(A/R)时,单核细胞与内皮细胞黏附及细胞间黏附分子-1(ICAM-1)mRNA表达并观察油茶皂苷C(SQS-C)对此的影响。方法:内皮细胞A/R前3h,分别加入终浓度为0.1,1.0和10.0μmol/LSQS-C,再A/R5h,比色法测定内皮细胞与单核细胞的黏附率,RT-PCR法检测内皮细胞ICAM-1mRNA的表达。结果:A/R时,单核细胞与内皮细胞的黏附显著增加(P<0.01);SQS-C能明显降低细胞黏附率,减少内皮细胞ICAM-1mRNA的表达(P<0.05),并呈剂量依赖性。结论:SQS-C能抑制缺氧/复氧损伤的内皮细胞与单核细胞的黏附,减少内皮细胞ICAM-1mRNA的表达。     

蜂胶乔松素对脂多糖诱导人脐静脉内皮细胞的保护作用

目的:观察蜂胶乔松素对脂多糖诱导人脐静脉内皮细胞的影响,探讨其对人脐静脉内皮细胞可能的保护作用。方法:实验于2006-03/10在泰山医学院生命科学研究所(省重点实验室)完成。①实验材料:取出生1h内新生儿脐带,患者知情同意。②实验分组及方法:培养人脐静脉内皮细胞,建立脂多糖损伤模型(以10mg/L的脂多糖培养液培养细胞12h),实验分为空白对照组(加等量D-Hank’s液)、脂多糖组(10mg/L)、乔松素组(加10mg/L脂多糖预孵育12h后,按50,100,200mg/L分别加入乔松素),各组设8个复孔,共同孵育24h。③实验评估:光镜下观察细胞形态,MTT法观察乔松素对人脐静脉内皮细胞活性的影响,ELISA方法检测培养上清中血管假血友病因子的含量,TUNEL检测细胞凋亡率。结果:①细胞形态:空白对照组细胞紧密贴壁,呈铺路石状生长。脂多糖组可见多数细胞呈圆形;乔松素组见上述细胞较脂多糖组明显减少。②乔松素对人脐静脉内皮细胞活性、凋亡及血管假血友病因子含量的影响:与对照组比较,脂多糖组能明显诱导人脐静脉内皮细胞的凋亡(P<0.01),不同浓度乔松素组可改善内皮细胞形态,组织活性明显升高(P<0.05),同时抑制内皮细胞血管假血友病因子的释放(P<0.05),使脂多糖诱导的人脐静脉内皮细胞凋亡细胞数明显减少(P<0.05)。结论:乔松素能增强人脐静脉内皮细胞活性,抑制脂多糖诱导人脐静脉内皮细胞的凋亡,从而发挥可能的内皮细胞保护功能。     

Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

Transient aplastic crisis in children with congenital hemolytic anemias has been linked epidemiologically to infection with a serum parvovirus-like virus (SPLV). The virus is found in the blood in the early stages of the crisis, and serum containing SPLV inhibits erythroid colony formation in vitro. After sedimentation of virus-containing sera through a sucrose density gradient, colony inhibitory activity is present in the particulate fraction and separate from serum immunoglobulins. No inhibitory activity can be recovered from convalescent-phase sera after similar fractionation procedures. Inhibition of erythroid colony formation in vitro is not a feature of sera from other viral infections. The pattern of resistance of SPLV activity to chemicals and enzymes is compatible with it being a parvovirus. By using replating techniques, a target of SPLV has been identified as a late erythroid progenitor cell. Neither SPLV antigen nor anti-SPLV IgM was present in the sera of patients with other forms of bone marrow failure.     

氧自由基对培养人脐静脉内皮细胞代谢产物的影响

目的：观察氧自由基对培养的人脐静脉内皮细胞产生的裂解不对称二甲精氨酸的酶--二甲精氨酸-二甲赖氨酸水解酶活性及表达的影响，以探讨不对称二甲精氨酸代谢机斜及卡托普利的作用。方法：实验于2003—05／2004—06在南昌大学医学分子中心进行。采用改良的Jaffe法培养人脐静脉内皮细胞，取生长良好的3～6代人脐静脉内皮细胞分为4组：①空白对照组：加DMEM培养液。②氧自由基0．01，0.1mmol／L组：分别加入氧自由基0.01，0.1mmol／L。③卡托普利组：同时加入氧自由基0.1mmol／L及卡托普利（100mg／L）共孵。孵育24h后，检测上清液中一氧化氮、一氧化氮合酶活性、内皮细胞的代谢产物不对称二甲精氨酸浓度以及反应二甲精氨酸-二甲赖氨酸水解酶酶活性的L-瓜氨酸浓度，采用Western blotting测定细胞裂解液中二甲精氨酸-二甲赖氨酸水解酶的蛋白表达。结果：①二甲精氨酸浓度：氧自由基0，01，0.1mmol／L组均高于空白对照组[（2．71&;#177;0．35），（4．78&;#177;0．67），（0．81&;#177;0．12）μmol／L，P〈0．05，0．01]，卡托普利组低于氧自由基0．1mmol／L组[（2．03&;#177;0．35）μmol／L．P〈0．01]。②L-瓜氨酸浓度：氧自由基0．01，0．1mmol／L组均低于空白对照组（P〈0．05，0．01）．卡托普利组高于氧自由基0．1mmol／L组（P〈0．01）。③一氧化氮浓度及一氧化氮合酶活性：氧自由基0．01，0．1mmol／L组均低于空白对照组（P〈0．05，0．01），卡托普利组高于氧自由基0．1mmol／L组（P〈0．01）。④二甲精氨酸-二甲赖氨酸水解酶的蛋白表达：各组间无差异（P〉0．05）。结论：氧自由基培养下，内皮损伤，不对称二甲精氨酸的增加与二甲精氨酸-二甲赖氨酸水解酶的活性减弱有关，与其蛋白的表达无关。而卡托普利则通过增加二甲精氨酸-二甲赖氨酸水解酶活性促进不对称二甲精氨酸代谢，使一氧化氮合酶活性增加，抑制氧自由基对内皮功能的损伤。     

氧自由基对培养人脐静脉内皮细胞代谢产物的影响

目的:观察氧自由基对培养的人脐静脉内皮细胞产生的裂解不对称二甲精氨酸的酶--二甲精氨酸-二甲赖氨酸水解酶活性及表达的影响,以探讨不对称二甲精氨酸代谢机制及卡托普利的作用。方法:实验于2003-05/2004-06在南昌大学医学分子中心进行。采用改良的Jaffe法培养人脐静脉内皮细胞,取生长良好的3～6代人脐静脉内皮细胞分为4组:①空白对照组:加DMEM培养液。②氧自由基0.01,0.1mmol/L组:分别加入氧自由基0.01,0.1mmol/L。③卡托普利组:同时加入氧自由基0.1mmol/L及卡托普利(100mg/L)共孵。孵育24h后,检测上清液中一氧化氮、一氧化氮合酶活性、内皮细胞的代谢产物不对称二甲精氨酸浓度以及反应二甲精氨酸-二甲赖氨酸水解酶酶活性的L-瓜氨酸浓度,采用Westernblotting测定细胞裂解液中二甲精氨酸-二甲赖氨酸水解酶的蛋白表达。结果:①二甲精氨酸浓度:氧自由基0.01,0.1mmol/L组均高于空白对照组[(2.71±0.35),(4.78±0.67),(0.81±0.12)μmol/L,P<0.05,0.01],卡托普利组低于氧自由基0.1mmol/L组[(2.03±0.35)μmol/L,P<0.01]。②L-瓜氨酸浓度:氧自由基0.01,0.1mmol/L组均低于空白对照组(P<0.05,0.01),卡托普利组高于氧自由基0.1mmol/L组(P<0.01)。③一氧化氮浓度及一氧化氮合酶活性:氧自由基0.01,0.1mmol/L组均低于空白对照组(P<0.05,0.01),卡托普利组高于氧自由基0.1mmol/L组(P<0.01)。④二甲精氨酸-二甲赖氨酸水解酶的蛋白表达:各组间无差异(P>0.05)。结论:氧自由基培养下,内皮损伤,不对称二甲精氨酸的增加与二甲精氨酸-二甲赖氨酸水解酶的活性减弱有关,与其蛋白的表达无关。而卡托普利则通过增加二甲精氨酸-二甲赖氨酸水解酶活性促进不对称二甲精氨酸代谢,使一氧化氮合酶活性增加,抑制氧自由基对内皮功能的损伤。     

Growth inhibitory effects of celecoxib in human umbilical vein endothelial cells are mediated through G1 arrest via multiple signaling mechanisms

Evidence suggests that the angiogenic endothelium represents an important target through which celecoxib mediates in vivo antitumor effects. Nevertheless, the pharmacologic basis for celecoxib-caused growth inhibition in endothelial cells in vitro remains to be defined. Previously, we showed that celecoxib-induced apoptosis in PC-3 prostate cancer cells was mediated in part through the inhibition of 3-phosphoinositide-dependent kinase-1/Akt signaling. Our present findings show that celecoxib inhibits the growth of human umbilical vein endothelial cells (HUVEC) with pharmacologic profiles reminiscent of those of PC-3 cells. The underlying antiproliferative mechanism, however, may differ between these two cell types considering differences in the functional status of many tumor suppressors, including PTEN, p53, and retinoblastoma, all of which play integral roles in regulating cell cycle progression and survival. From a mechanistic perspective, the genomic integrity of the HUVEC system presents a vastly different intracellular context to examine how celecoxib acts to induce growth inhibition. Here, we obtain evidence that the antiproliferative effects of celecoxib and its close, cyclooxygenase-2-inactive analogue 4-[5-(2,5-dimethylphenyl)-3(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (DMC) in HUVECs at pharmacologically attainable concentrations (10-20 micromol/L) are attributable to the inhibition of phosphoinositide-dependent kinase-1/Akt signaling and cyclin-dependent kinase. Especially, celecoxib- and DMC-mediated G1 arrest is associated with attenuated retinoblastoma phosphorylation through the inhibition of multiple cyclin-dependent kinases (IC50, 10-35 micromol/L). Moreover, both celecoxib and DMC reduce neovascularization in the chicken chorioallantoic membrane assay, suggesting the involvement of a cyclooxygenase-2-independent mechanism in the in vivo antiangiogenic effects of celecoxib.     

尾加压素Ⅱ对人脐静脉内皮细胞释放一氧化氮和前列环素的影响

目的:观察不同浓度的尾加压素Ⅱ对人脐静脉内皮细胞释放一氧化氮、前列环素,以及对一氧化氮合酶活性的影响,进而探讨尾加压素Ⅱ舒张血管的机制。方法:实验于2005-04/08在武汉大学人民医院老年病科实验室完成。根据所加入尾加压素Ⅱ的浓度不同(10-9～10-7mol/L),将体外培养的人脐静脉内皮细胞随机分为4组:空白对照组、10-9mol/L组、10-8mol/L组及10-7mol/L组。干预24h后,用化学比色法检测细胞培养上清液中NO2-,NO3-的水平及一氧化氮合酶的活性,用放射免疫法检测碘[125Ⅰ]-6-酮前列腺素F1α的水平。结果:①一氧化氮水平:后三组与空白对照组相比,NO2-,NO3-的含量呈浓度依赖性增加,其中10-7mol/L浓度组显著高于空白对照组(21.37±2.21,16.51±1.33,P<0.01)。②一氧化氮合酶活性:后三组与空白对照组相比,一氧化氮合酶呈浓度依赖性增加,其中10-7mol/L浓度组显著强于空白对照组(70.65±15.63,36.62±11.16,P<0.01)。③前列环素水平:后三组与空白对照组相比,6-酮前列腺素F1α含量呈浓度依赖性增加,其中10-7mol/L浓度组显著高于空白对照组(572.69±1.86,563.93±2.66,P<0.01)。结论:尾加压素Ⅱ能刺激人脐静脉内皮细胞释放一氧化氮及前列环素,提示尾加压素Ⅱ可能通过刺激一氧化氮和前列环素的产生而发挥舒张血管的作用。     

Relationship between erythropoietin and nitric oxide in the contraction of rat renal arcuate arteries and human umbilical vein endothelial cells.

We have investigated the effects of recombinant human erythropoietin (EPO) on the responses of rat renal arcuate arteries to dopamine, noradrenaline and acetylcholine and on the release of NO from human umbilical vein endothelial cells (HUVEC) in culture. Noradrenaline induced a concentration-dependent constriction and acetylcholine a concentration-dependent relaxation of the vessels. The effects of dopamine were concentration-dependent, leading to relaxation of the vessels at low concentrations and contraction of the vessels at high concentrations. N(G)-Nitro-L-arginine methyl ester (L-NAME; 0.1 mM) did not change the vasoconstrictor responses to noradrenaline and dopamine, but inhibited the acetylcholine- and dopamine-induced vasorelaxation. Neither 0.1 nor 20 units.ml(-1) EPO affected noradrenaline-induced constriction, or dopamine- or acetylcholine-induced relaxation, of the vessels. EPO at 20 units. ml(-1) attenuated dopamine-induced constriction of the vessels. This effect was blunted by application of L-NAME, suggesting that EPO may stimulate dopamine-mediated NO release from these vessels. EPO stimulated NO release from the resting HUVEC in a concentration- and time-dependent manner, an effect that was inhibited by the presence of N(G)-nitro-L-arginine. These data suggest that, in vitro, EPO is able to stimulate NO release from rat renal arcuate arteries and HUVEC in culture. Whether these acute short-term actions can be related to the longer-term actions of EPO remains to be resolved.     

三种阳离子脂质体介导血管内皮细胞基因转染效率的比较

目的:评价不同阳离子脂质体介导基因转染血管内皮细胞的转染效率。方法:实验于2006-12/2007-02在中山大学生化实验室及广州市创伤外科研究所完成。采用增强型绿色荧光蛋白基因为报告基因,分别采用Lipofectin、Lipofectamine、Dosper3种不同的阳离子脂质体为载体,转染人脐静脉血管内皮细胞。在24孔板中,每孔加入人脐静脉血管内皮细胞悬液(1×106个细胞),各孔分别加入3种不同阳离子脂质体增强型绿色荧光蛋白质粒复合物,分别于培养24,48h后用荧光显微镜及流式细胞仪测定增强型绿色荧光蛋白在细胞内的表达及转染效率。结果:3种不同阳离子介导的增强型绿色荧光蛋白基因转染的人脐静脉血管内皮细胞内均有绿色荧光蛋白表达,24h后明显,48h后达高峰。Dosper介导组绿色荧光细胞百分比明显高于Lipofectin介导组及Lipofectamine介导组,差异有非常显著意义(P<0.01)。结论:Dosper介导的血管内皮细胞基因转染效率较高,较适合作为血管内皮细胞的基因转染载体。     

Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells

Summary.  The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn²⁺-dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn²⁺-dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn²⁺-dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.     

抑制胞浆型磷脂酶A2活性对内毒素诱导人脐静脉内皮细胞分泌瘦素的影响

目的 通过改变胞浆型磷脂酶A2(cPLA2)的活性,检测细菌脂多糖(LPS)及Ca2+载体A23187诱导的人脐静脉内皮细胞株(ECV-304)上清液中瘦素(Leptin)水平的变化,探讨在体外炎症状态下cPLA2活性与细胞分泌Leptin的关系.方法 体外培养ECV-304细胞.实验1:将细胞分为空白对照组,LPS 3个浓度5、10、20 μg/ml刺激组,Ca2+载体A23187 3个浓度0.1、 1.0、10.0 μmol/L刺激组共7个组,分别作用6、12、24 h后收集上清液.实验2:根据实验1结果将细胞分为空白对照组,LPS 20 μg/ml刺激组,cPLA2特异性抑制剂AACOCF3 3个浓度0.1、1.0、10.0 μmol/L与LPS合用刺激组,丝裂素活化蛋白激酶上游激酶1/2(MEK1/2)抑制剂 U0126 3个浓度0.1、1.0、5.0 μmol/L与LPS合用刺激组共8个组,在LPS刺激前1 h加入AACOCF3或U0126,LPS刺激24 h后收集上清液.采用放射免疫分析法检测Leptin水平.结果 实验1:随LPS刺激浓度增加和时间延长,细胞释放Leptin浓度逐渐减少,LPS 20 μg/ml组作用24 h后Leptin浓度(ng/ml)较空白对照组显著下降(0.540±0.109比0.823±0.048,P<0.05).但A23187对细胞分泌Leptin并无显著影响.实验2:LPS刺激能使细胞分泌Leptin浓度(ng/ml)明显下降(0.558±0.069比0.825±0.067,P<0.05);而用不同浓度AACOCF3或U0126干预后,细胞分泌Leptin的浓度(ng/ml)有所回升,且呈浓度依赖性(AACOCF3 0.1、1.0、10.0 μmol/L组分别为0.673±0.135、 0.723±0.055、 0.797±0.062;U0126 0.1、 1.0、5.0 μmol/L组分别为0.698±0.112、 0.862±0.184、0.935±0.145),AACOCF3 1.0 μmol/L、10.0 μmol/L组和U0126 1.0 μmol/L、5.0 μmol/L组Leptin浓度均显著高于LPS 20 μg/ml刺激组(均P<0.05).结论 在由LPS诱导的体外炎症状态下,Leptin的分泌与cPLA2的活性具有一定的关系.

Abstract：

Objective To determine Leptin levels in supernatant fluid of culture of human umbilical vein endothelial cells (ECV-304) after being challenged by lipopolysaccharide (LPS) and calcium ion vector A23187, and to explore the possible relation between Leptin release and cytosolic phospholipase A2 (cPLA2) activity in an inflammatory cell model. Methods ECV-304 cells were cultured in vitro. Experiment 1: the cells were divided into seven groups: blank control group, LPS 5, 10, 20 μg/ml stimulation groups, A23187 0.1, 1.0, 10.0 μmol/L stimulation groups. The supernatants were collected at 6, 12 and 24 hours. Experiment 2: according to the results of experiment 1, the cells were divided into eight groups: blank control group, LPS 20 μg/ml stimulation group, the inhibitor of cPLA2 AACOCF3 0.1, 1.0, 10.0 μmol/L plus LPS stimulation groups, the inhibitor of mitogen-activated protein/extracellular signal-regulated protein kinase kinase 1/2 (MEK1/2) U0126 0.1, 1.0, 5.0 μmol/L plus LPS stimulation groups, with AACOCF3 or U0126 added 1 hour before the addition of LPS, and the supernatants were collected 24 hours after the addition of LPS. Leptin level was determined by radioimmunoassay. Results Experiment 1: with increase in LPS concentration and prolongation of time, Leptin release was decreased gradually. After 24 hours of interaction the concentration of Leptin (ng/ml) in LPS 20 μg/ml group was decreased significantly compared with the blank control group (0.540±0.109 vs. 0.823±0.048, P<0.05). However, A23187 had no significant effect on Leptin release. Experiment 2: LPS rendered cells to release less Leptin (ng/ml: 0.558±0.069 vs. 0.825±0.067, P<0.05); by adding AACOCF3 or U0126 in different concentration before adding LPS rendered the cells to release more Leptin (ng/ml), and it showed concentration-dependent (the AACOCF3 0.1, 1.0, 10.0 μmol/L groups were 0.673±0.135, 0.723±0.055, 0.797±0.062, respectively; the U0126 0.1, 1.0, 5.0 μmol/L groups were 0.698±0.112, 0.862±0.184, 0.935±0.145, respectively). The release of Leptin in AACOCF3 1.0 μmol/L, 10.0 μmol/L and U0126 1.0 μmol/L, 5.0 μmol/L groups was significantly higher than LPS 20 μg/ml stimulation group (all P<0.05). Conclusion There is a possible relation between Leptin release and cPLA2 activity in inflammatory cells induced by LPS.     

人脐静脉内皮细胞血管紧张素转化酶及血管紧张素转化酶2表达与氧化型低密度脂蛋白的关系

目的:新近研究发现血管紧张素转化酶2与血管紧张素转化酶在功能上相拮抗.采用反转录-聚合酶链反应及蛋白质免疫印迹方法,观察氧化型低密度脂蛋白对人血管内皮细胞血管紧张素转化酶及血管紧张素转化酶2表达的影响.方法:实验于2007-05/08在南方医科大学附属珠江医院中心实验室完成.①用正常人新鲜血浆制备氧化低密度脂蛋白,人脐静脉内皮细胞株复苏、传代后取生长良好的3～6代用于实验.②实验分为7组:空白对照组加无血清培养基;20,40,80mg/L氧化型低密度脂蛋白组分别与人脐静脉内皮细胞孵育24h;40mg/L氧化型低密度脂蛋白分别与人脐静脉内皮细胞孵育6,12,24h.各组实验重复6次.③提取细胞总RNA及蛋白,应用反转录-聚合酶链反应检测血管紧张素转化酶、血管紧张素转化酶2mRNA表达水平;以蛋白质免疫印迹法检测血管紧张素转化酶、血管紧张素转化酶2蛋白表达水平.结果:①与空白对照组比较,氧化低密度脂蛋白20,40,80mg/L组血管紧张素转化酶mRNA表达增加,血管紧张素转化酶2mRNA表达降低(P＜0.01),不同剂量组间差异亦有显著性意义(P均＜0.01).40mg/L氧化型低密度脂蛋白6,12,24h组血管紧张素转化酶mRNA表达增加,血管紧张素转化酶2mRNA表达减少(P＜0.05),不同时间点间差异亦有显著性意义(P均＜0.05).②与空白对照组比较,氧化低密度脂蛋白20,40,80mg/L组血管紧张素转化酶蛋白表达增加,血管紧张素转化酶2蛋白表达减少(P＜0.01),不同剂量组间差异亦有显著性意义(P均＜0.01).40mg/L氧化型低密度脂蛋白6,12,24h组血管紧张素转化酶蛋白表达增加,血管紧张素转化酶2蛋白表达降低(P＜0.05),不同时间点间差异亦有显著性意义(P均＜0.05).结论:氧化型低密度脂蛋白可上调血管紧张素转化酶蛋白及基因表达,下调血管紧张素转化酶2蛋白及基因表达,且呈剂量和时间依赖性.