降钙素基因相关肽对血管内膜损伤后内皮细胞功能恢复的影响

在球囊剥脱大鼠主动脉内膜造成的内皮损伤模型上,用ＣＧＲＰ（１０μｇ／ｋｇ／ｄ）治疗后观察血管内皮合成释放ｖＷＦ含量及ｔ－ＰＡ活性的变化。结果表明ＣＧＲＰ能明显降低ｖＷＦ含量和增加ｔ－ＰＡ活性。提示ＣＧＲＰ能减轻内皮损伤反应,在促内皮细胞增殖的同时对内皮某些功能恢复亦有一定的作用     

睾酮对人血管内皮细胞产生NO、tPA和PAI-1的影响

目的:观察不同浓度睾酮对人血管内皮细胞生长、产生舒张因子及纤溶活性的影响.方法:体外培养人脐静脉内皮细胞(HUVEC),分为五个浓度睾酮组及单纯培养基对照组.做MTT实验观察睾酮对HUVEC生长的影响;还原酶法测定各组HUVEC释放NO量;ELISA法测定各组培养基中纤溶酶原激活物(tPA)及其抑制物(PAI-1)含量.结果:3×10-10mol/L-3×10-8mol/L睾酮组与对照组相比细胞生长良好,无明显差别;而大于生理剂量的两组(3×10-6～3×10-1mol/L)3 d后细胞生长明显受到抑制(P＜0.05).各浓度睾酮组产生NO量与对照组无明显区别.而3×10-10 mol/L～3×10-8 mol/L睾酮组tPA含量明显高于对照组(P＜0.01);大剂量组tPA产生明显减少(P＜0.01).所有实验组的PAI-1含量均明显降低.结论:生理及略低于生理剂量的睾酮对HUVEC生长及释放NO无不利影响,且增加纤溶活性.说明生理剂量睾酮对血管内皮功能、心血管系统有一定的保护作用,有利于防止动脉粥样硬化的发生、发展.     

同型半胱氨酸对人脐静脉血管内皮细胞tPA 及PAI-1基因表达的影响

目的探讨同型半胱氨酸(Hcy)对纤溶系统的影响,观察Hcy在转录水平对人脐静脉血管内皮细胞(HUVEC)表达组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制剂1(PAI1)的影响。方法将体外培养的HUVEC分为生理浓度(10μmol/LHcy)组,病理浓度(50、200、500μmol/L)Hcy组及单纯培养基组(0μmol/LHcy),培养24h后,提取RNA,反转录聚合酶链反应分析(RTPCR)法分析各组tPA及PAI1基因表达水平。结果500μmol/LHcy组与10μmol/LHcy组相比,tPAmRNA基因表达明显下调(P<0.05),PAI1mRNA表达则明显上调(P<0.05)。而与单纯培养基组相比,10μmol/LHcy组tPAmRNA表达明显增高(P<0.05)。结论生理浓度Hcy可以增加纤溶系统活性,减少血栓性疾病的发生。高Hcy(病理浓度)则抑制纤溶系统活性,促进缺血性心脑血管疾病的发生。     

叶下珠有效部位的溶栓作用及其对PAI-1和tPA活性的影响

采用改良的Charlton和Tomihisa等方法评价叶下珠植物(Phyllanthus urinaria)含corilagin的水溶性有效部位(代号PUW)对电刺激大鼠颈动脉血栓的溶栓作用;应用发色底物方法测定PUW在体内外对血浆tPA、血浆或血小板释放的PAl-1活性的影响。结果显示,5mg／kg的PUW,其再通率为50％,再栓率为60％;10mg／kg PUW的血管再通率为60％,其再栓率为33．3％,低于2万U／kg尿激酶42．9%的再栓率。再通后1h内,血管开放状态表现为,2万U／kg尿激酶组的血管开放状态与5mg／kg PUW组的相似;10mg／kg PUW组,其持续再通率高于2万U／kg的尿激酶组。PUW在体外或静脉注射均明显降低血浆PAI—1活性,同时提高血浆tPA的活性;PUW静注还明显抑制血小板释放的PA1-1活性。本实验结果提示,PUW静脉注射显著提高闭塞颈动脉的再通率,同时降低再通后颈动脉的再栓率;抑制PAI—1活性,同时提高tPA的活性可能是PUW具有较好溶栓作用的分子机制。     

槲皮素对过氧化氢所致PC-12细胞氧化损伤的保护作用

目的:研究槲皮素(quercetin)是否对过氧化氢所致PC-12细胞氧化损伤具有保护作用,以及可能的保护机制。方法:用PC-12细胞建立H2O2氧化损伤模型;测定SOD、T-AOC生化指标判断细胞抗氧化能力;半定量RT-PCR法检测糖皮质激素受体(GR)基因转录水平。结果:①MTT结果:H2O2能使细胞活力显著降低(P0.01),槲皮素的预孵处理能够明显减轻H2O2对PC-12细胞的氧化损伤(P0.050.01)。②SOD活性结果:H2O2作为氧化损伤因素使得细胞SOD活性应激上升(P0.01),Qu组细胞SOD活性维持在较低水平(P0.01)。③T-AOC结果:槲皮素能够显著提高PC-12细胞的总抗氧化能力(P0.01)。④半定量RT-PCR结果:H2O2使PC-12细胞GR基因转录水平显著降低,槲皮素能够减轻其转录所受影响(P0.01)。结论:槲皮素与PC-12细胞的共孵育,提高了细胞整体的抗氧化能力,维持了GR基因的转录水平,继而保护细胞免受后续H2O2的氧化损伤和炎症反应,维持细胞生化环境的稳态。     

槲皮素对血管内皮细胞增殖和迁移的抑制作用

本实验研究了槲皮素对人脐静脉内皮细胞株(ECV304)增殖和迁移的抑制作用。研究发现,槲皮素作用一定的时间后,能明显抑制ECV304细胞的增殖(IC50为50．08μg/ml)和迁移(IC50为7．84μg／ml),而且其抑制作用呈浓度依赖性;细胞出现凋亡形态学改变,琼脂糖凝胶电泳形成DNA条带,推测其诱导细胞发生了凋亡。     

三七皂苷Rg1对tPA和PAI-1活性的调节作用

探讨三七皂苷Rg1对组织型纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物(PAI-1)活性的调节作用。运用发色底物方法测定三七皂苷Rg1在体外和静脉注射对家兔血浆纤溶酶原激活物(tPA)和血浆或血小板释放的纤溶酶原激活物抑制物(PAI-1)水平的影响。结果表明,三七皂苷Rg1在体外呈浓度依赖性明显抑制血浆PAI-1活性,同时提高血浆tPA活性;30和60 mg/kg的三七皂苷Rg1静脉注射显著抑制血浆PAI-1活性,提高血浆tPA活性,同时降低凝血酶激活的血小板所释放的PAI-1水平。本实验提示三七皂苷Rg1能抑制PAI-1活性,同时升高tPA活性可能是其抗血栓作用的分子机制之一。     

纤溶酶原Kringle 5的抑制血管增生作用

纤溶酶原Kringle 5是纤溶酶原中与血管抑素相连的另一个联环结构域,它能抑制内皮细胞的增殖、迁移,并能诱导内皮细胞凋亡和细胞周期停滞,具有很强的抑制血管生成活性,是抑制活性最强的纤溶酶原水解片段。同时它具有分子量小、性质稳定、毒副作用小、特异性高等优点,是一个有潜在临床应用价值和开发前景的治疗血管增生性疾病的药物。     

槲皮素对培养内皮细胞释放前列环素的影响

应用放射免疫分析方法,研究了槲皮专对体外培养的人脐静脉内皮细胞释放前列环素的影响。实验表明：活化血小板与内皮细胞共同孵育１０,３０和１２０ｍｉｎ后,内皮细胞条件培养液中６一酮一ＰＧＦ１ｍ量显著增加（ｐ＜０．０ｌ）。槲皮素１,５和２０ＩＬｌｎｏｌ。－１与内皮细胞预孵１０ｍｉｎ,可抑制活化血小板引起的内皮细胞６一酮一ＰＧＦ１ｍ的大量释放。对于正常的内皮细胞,树皮素则增加６一酮一ＰＧＦ１ａ的基础释放量。     

rt-PA治疗急性脑梗死的近期临床疗效及其影响因素分析

目的:探讨重组组织型纤溶酶原激活剂(recombinant tissue plasminogen activator,rt-PA)溶栓治疗急性脑梗死的近期临床疗效及其影响因素。方法:回顾性分析2013年8月至2015年1月于我院神经内科接受rt-PA溶栓治疗的88例急性脑梗死患者的临床资料,分析患者入院及发病24h的美国国立卫生研究院卒中量表(NIHSS)评分,以评分的差值作为结局变量,分为预后良好的观察组(NIHSS差值≥4分或≥50%)及预后不良的对照组(NIHSS差值4分或50%)。选取溶栓前临床及实验室资料,筛选不良预后的可能影响因素,并进行单因素及多因素分析。结果:88例患者溶栓治疗后近期预后良好者46例(52.27%),预后不良者42例(47.73%)。4.5 h内接受溶栓者好转率为57.14%(36/63),4.5 h接受溶栓者好转率为32.00%(8/25),显著低于4.5h内接受溶栓者,差异具有统计学意义(P0.05)。单因素分析结果显示糖尿病史、高密度脂蛋白、血糖、溶栓时间窗、NIHSS评分变量是影响rt-PA溶栓疗效的因素,进一步多因素分析结果显示溶栓时间窗、血糖、NIHSS评分是影响rt-PA溶栓疗效的因素。结论:rt-PA静脉溶栓治疗急性脑梗死的近期疗效有明显的时间依赖性,且血糖、NIHSS评分为早期预后的影响因素。     

Differential effects of soluble and immobilized fibronectins on aortic endothelial cell proliferation and attachment

Summary We studied the effects of soluble and immobilized forms of plasma fibronectin on bovine aortic endothelial cell (AEC) proliferation and attachment. Soluble fibronectin stimulated AEC growth at 10 μg/ml, but at higher concentrations of soluble fibronectin AEC growth was progressively inhibited. The growth rates of arterial smooth muscle cells (ASMC) and dermal fibroblasts (DF) were not altered by soluble fibronectin concentrations of 10 to 100 μg/ml. Plasma fibronectin, immobilized by attachment to culture dish surfaces, had no significant effects on the proliferation of any of the cell types examined. The attachment rates of AEC were decreased in the presence of 50 μg/ml soluble fibronectin. Immobilized fibronetin increased the rate of AEC attachment, but had no significant effects on ASMC or DF attachment; however, 12 h after plating there was nearly 100% attachment in all groups, whether or not fibronectin was present in the system. That soluble and immobilized fibronectins elicit disparate cellular responses is consistent with published reports of different cell surface receptors for different forms of the protein; in this manner, cells enmeshed in an interstitial matrix containing immobilized fibronectin could still respond to soluble fibronectin in the extracellular milieu. These studies were supported in part by grant EY-0229 from the National Institutes of Health, Bethesda, MD.     

肌肽对低氧所致大鼠血管内皮细胞损伤的保护作用

目的：研究肌肽对低氧所致大鼠血管内皮细胞损伤的影响。方法：建立低氧条件下大鼠血管内皮细胞损伤模型,用MTT法观察肌肽对低氧损伤的血管内皮细胞活性的影响,测定细胞培养基中LDH活力,并对细胞骨架进行考马斯亮蓝R-250染色观测其细胞结构。结果：浓度为10mmol/L～20mmol/L肌肽孵育血管内皮细胞6h后,可以抑制缺氧12h和24h引起的血管内皮细胞活性下降,同时减少LDH的释放,保持细胞骨架完整。结论：肌肽对低氧所致的血管内皮细胞损伤具有保护作用。     

Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10⁴ HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 10⁴. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 10⁴. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

雌激素处理的hBMSC对高糖诱导血管内皮细胞损伤的保护作用*

目的: 探讨雌激素处理人骨髓间充质干细胞(hBMSC)对高糖诱导的人脐静脉血管内皮细胞(HUVEC)损伤的保护作用及机制。方法: 采用30 mmol/L葡萄糖刺激hBMSC细胞建立高糖模型并分组:以无刺激者为高糖对照组(HG组)、以20 μmol/L雌激素处理者为高糖雌激素组(HG+E2组)、以5 μmo/L蛋白激酶B(PKB/Akt)抑制剂Triciribine预处理45 min后,再以20 μmol/L雌激素处理者为高糖Akt抑制剂组(HG+E2+Triciribine组)和正常条件培养的hBMSC为正常对照组(NG组)。分别于处理12 h后,采用CCK8法检测各组hBMSC的细胞活力,硝酸还原酶法和ELISA法检测各组培养基上清中NO、VEGF和IL-8的含量(n=6),48 h后采用Western blot检测内皮型一氧化氮合酶(eNOS)和磷酸化eNOS(p-eNOS)蛋白表达水平(n=3)。此外,提取各组hBMSC的培养基上清作为条件培养基(CM)培养人脐静脉血管内皮细胞(HUVEC)并分组为:HG-CM组(HG组条件培养基处理)、HG+E2-CM组(HG+E2组条件培养液处理)、HG+E2+Triciribine-CM组(HG+E2+Triciribine组条件培养基处理)和HG-H组(高糖对照组,30 mmol/L葡萄糖终浓度处理),分别于12 h后,采用CCK8法检测各组HUVEC的细胞活力(n=6),24 h后采用流式细胞术检测各组HUVEC细胞的凋亡率(n=3);48 h后采用划痕实验观察各组HUVEC细胞的迁移率(n=3)。结果: 与NG组相比,HG组中hBMSC细胞活力和细胞内eNOS蛋白磷酸化水平降低(P<0.05),细胞培养液上清中NO、VEGF和IL-8含量减少(P<0.05);与HG组相比,HG+E2组中hBMSC的细胞活力和细胞中eNOS蛋白磷酸化水平显著增加(P<0.05),细胞培养基上清中NO、VEGF和IL-8含量增加(P<0.05),而当hBMSC细胞中Akt蛋白活性被抑制后,HG+E2+Triciribine组中上述结果指标呈反向变化(P<0.05)。此外,与HG-CM组相比,HG+E2-CM组中HUVECs的细胞活力和迁移能力显著增加(P<0.05),细胞凋亡比例降低(P<0.05),而与HG+E2-CM组相比,HG+E2+Triciribine-CM组中HUVECs的细胞活力和迁移能力降低(P<0.05),细胞凋亡比例增加(P< 0.05)。结论: 雌激素可能通过激活hBMSC细胞Akt/eNOS信号通路,促进NO、VEGF和IL-8的分泌,进而增加HUVECs的细胞活力和迁移能力,并抑制细胞凋亡的发生,对高糖诱导的HUVECs细胞损伤发挥保护作用。     

Metabolic responses induced by thrombin in human umbilical vein endothelial cells

Metabolic responses induced by thrombin in human umbilical vein endothelial cells (HUVECs) were investigated by using the cytosensor technique. Thrombin increased the extracellular acidification rate of endothelial cells, measured as an index of metabolic activity with a cytosensor microphysiometer, in a concentration-dependent fashion with an EC(50) of 1.27+/-0.59 IU/ml, which was abolished by the MAP kinase inhibitor PD98059. When intracellular Ca(2+) was chelated or PKC was inactivated, PD98059 failed to abolish the thrombin-induced acidification rate response in HUVECs. In addition, the tyrosine kinase inhibitor genistein, PKC inhibitor calphostin C, and Na(+)/H(+)exchanger antagonist MIA also partly inhibited thrombin-induced acidification rate responses. It is suggested that thrombin stimulated rapid metabolic responses via MAP kinase in HUVECs, which are calcium- and PKC-dependent.     

Growth of human vascular endothelial cells on various types of microcarriers

Various types of microcarriers were tested as growth substrate for the cultivation of either endothelial cells from human umbilical cord veins or of EA. hy926, an immortalized cell line of endothelial origin. Cell growth was tested on microcarriers in tissue culture flasks and spinner flasks. Solid (Cytodex type I, II, III, Gelibeads, Mica) and macroporous (Polyhipe, CultiSpher GL, PolyporE type I) microcarriers were tested. For the solid carriers the best results were obtained with Mica and for the macroporous carriers with CultiSpher GL.Abbreviations DAPI 4,6-diamidino-2-phenylindole-di-hydrochloride - DEAE diethylaminoethyl - EC vascular endothelial cells - FGF fibroblast growth factor - HUVEC vascular endothelial cells from human umbilical cord veins - IF 11 mixture of Iscove's MDM and F12 basal media - NCS newborn calf serum - PBS phosphate buffered saline - TE 0.05% (w/v) trypsin, 0.02% (w/v) EDTA in PBS     

Effect of scavengers of active oxygen species and pretreatment with acetyl-salicylic acid on the injury to cultured endothelial cells by thrombin-stimulated platelets

Summary Thrombin-stimulated human platelets adhere to and injure cultured human endothelial cells. We hypothesize that generation of active oxygen species by the stimulated platelets are involved in the injury. To confirm this, catalase [final concentration (8.25 μg/ml)], superoxide dismutase (SOD) (10 μg/ml), ofd-mannitol (9 mg/ml) were added to the cell culture medium before the experiments. Platelet suspension (200.000/μl) and thrombin (4 U/ml) were added and the culture dishes shaken for 15 min at room temperature. In separate experiments the endothelial cells were pretreated with acetylsalicylic acid (0.05, 0.1, or 0.5 mM) to test whether the arachidonic acid metabolism of the endothelial cells is involved in the injury process. In preliminary experiments we were able to confirm that platelets, when stimulated by thrombin, produce chemiluminescence which was suppressed by mannitol but not by catalase or SOD. The degree of injury to cultured endotheial cells by thrombin-stimulated platelets, as measured by release of⁵¹Cr from prelabeled endothelial cells, was reduced significantly with the presence of mannitol, but only moderately when catalase or SOD had been added. Morphometric quantification based on scanning electron micrographs of the endothelial cells after exposure to thrombin-stimulated platelets in the presence of catalase or mannitol showed a reduced number of injured cells. Pretreatment of the endothelial cells with acetylsalicylic acid did not cause any significant change in the degree of endothelial cell injury as measured by the⁵¹Cr release. It is concluded that active oxygen species, in particular hydroxyl radicals, may be generated during thrombin stimulation of platelets and cause injury to the endothelial cells. This work was supported by the Norwegian Research Council for Science and the Humanities and the Norwegian Council for Cardiovascular Diseases. We express our gratitude for their grants.     

A serum-free medium formulation supporting growth of human umbilical cord vein endothelial cells in long-term cultivation

A serum-free medium formulation – TUD-1 – was developed supporting growth of HUVEC in tissue culture. Special features of the basal medium formulation are highly elevated levels of glutamine and serine as well as the inclusion of N-acetylcysteine and phosphoascorbic acid. The cellular mitogenic needs are satisfied by bFGF, VEGF, EGF and liver growth factor. Further hormone supplementation consists of insulin and hydrocortisone. A protocoll for serum-free passage of HUVEC was established for serum-free long-term cultivation of freshly isolated HUVEC for up to 20 cumulative population doublings without significant differences in final cell density compared to controls cultivated with serum. This revised version was published online in July 2006 with corrections to the Cover Date.