LPS促进HUVECs表达纤溶酶原激活物抑制物1

目的：观察LPS对脐静脉血管内皮细胞(HUVECs)表达组织纤溶酶原激活物(tPA)和纤溶酶原激活物抑制物1(PAI-1)的影响。 方法： 用生长良好的第2、3代HUVECs进行试验。用cell counting kit-8(CCK-8)测定LPS刺激后细胞活性变化；发色底物法测定LPS组和对照组培养液中tPA, PAI-1活性；RT-PCR检测细胞内tPA和PAI-1 mRNA水平。 结果： 与对照组相比，LPS(10 mg/L)对细胞活性没有明显差异。LPS诱导PAI-1活性在24-72 h显著升高(P<0.05),且显著上调PAI-1 mRNA，24 h达到峰值，以后渐降，72 h达到正常水平。而LPS组与对照组tPA活性与tPA mRNA无明显差异(P>0.05)。 结论： LPS(10 mg/L)可显著上调PAI-1 mRNA转录和分泌而不影响tPA mRNA，结果提示LPS可活化内皮细胞，诱发PAI-1 mRNA表达和蛋白分泌而抑制纤溶系统，这有利于微血栓的形成、血栓稳定,血液凝固和DIC发生。     

睾酮对人血管内皮细胞纤溶活性影响及机制

目的：观察睾酮对人血管内皮细胞分泌纤溶酶原激活物（tPA）、纤溶酶原激活物抑制物1（PAI-1）的影响及其机制。方法： 将体外培养的人血管内皮细胞（HUVEC）分为5个浓度睾酮组及单纯培养基对照组，MTT实验观察睾酮对细胞生长及活性影响。ELISA 法测各组tPA、 PAI-1含量。用雄激素受体拮抗剂(flutamide)预处理细胞后重复实验。结果： 生理及略低于生理剂量睾酮（3×10-10 mol/L-3×10-8 mol/L）可明显促进tPA 分泌（P＜0.01）；而大剂量则使tPA 含量明显减少（P＜0.01）。各睾酮组PAI-1含量均明显低于对照组（P＜0.05）。Flutamide 能有效消除睾酮的上述作用。结论： 生理浓度睾酮通过雄激素受体促进tPA分泌，降低PAI-1浓度而增强纤溶系统活性，有利于防止血栓性疾病的发生。     

体外培养的血管内皮细胞低氧低糖损伤后tPA、PAI-1表达变化

目的：观察体外培养的血管内皮细胞低氧低糖损伤后组织型纤溶酶原激活剂(tPA)、Ⅰ型纤溶酶原激活物抑制因子(PAI-1)表达变化，探讨脑缺血后纤溶系统的变化及机制。材料和方法：制备体外内皮细胞低氧低糖损伤模型，利用HE染色、免疫细胞化学染色观察tPA、PAI-1表达变化。结果：低氧低糖损伤后，tPA、PAI-1表达均明显增强。结论：成功制备体外内皮细胞低氧低糖损伤模型。内皮细胞低氧低糖损伤可以诱导tPA、PAI-1表达增多，进一步说明脑缺血损伤后tPA、PAI-1表达增加并参与损伤过程。     

胰岛素和葡萄糖对缺氧血管内皮细胞分泌tPA和PAI-1抗原的影响

目的: 观察胰岛素和葡萄糖对缺氧血管内皮细胞分泌组织型纤溶酶原激活物(tPA)及其抑制物-1(PAI-1)的影响。 方法:培养人脐静脉内皮细胞株ECV-304。分组实验:(1)常氧组;(2)在缺氧条件下又分为Ⅰ:缺氧对照组;Ⅱ:低浓度组:胰岛素150 mU/L、葡萄糖5.5 mmol/L;Ⅲ:中浓度组:胰岛素450 mU/L、葡萄糖15 mmol/L;Ⅳ:高浓度组:胰岛素900 mU/L、葡萄糖30 mmol/L;Ⅴ:渗透压对照组:甘露醇24.5 mmol/L。取培养4、8、12 h 3个时点,用ELISA法测定细胞培养上清液tPA、PAI-1抗原。结果:缺氧明显增加tPA和PAI-1抗原分泌,tPA/PAI-1值明显增加(P＜0.01)。胰岛素和葡萄糖能够刺激缺氧内皮细胞分泌tPA和PAI-1抗原,在缺氧8 h以内,tPA/PAI-1比值明显增加(P＜0.05)。随缺氧时间的延长,tPA/PAI-1值逐渐下降。结论:胰岛素和葡萄糖能够刺激内皮细胞分泌tPA和PAI-1抗原,在缺氧8 h以内,tPA/PAI-1值升高,纤溶活性升高,有利于局部自发性纤溶的发生。此作用可能是IGK治疗急性心肌梗死的机制之一。     

同型半胱氨酸对血管内皮细胞PAI-1活性及其mRNA水平的影响

探讨血浆同型半胱氨酸（Hcy）致血管病变的机制。培养人脐静脉内皮细胞株，用发色底物法测定细胞上清的纤溶酶原激活物抑制物－1（PAI－1）活性，细胞原位杂交及辉度扫描检测PAI－1 mRNA水平。结果表明H 驻作用血管内皮细胞后，PAI－1活性随Hcy浓度增加和Hcy作用时间延长而呈递增趋势，PAI－1 mRNA灰度面积积分值随Hyc浓度的增加而增加。提示Hcy抑制内皮细胞纤溶能力是Hcy致血栓形成和血管病变的一个机制。     

同型半胱氨酸和叶酸对内皮细胞tPA合成及其mRNA表达的影响

目的探讨同型半胱氨酸(Hcy)及叶酸对内皮细胞纤溶系统的作用,观察Hcy和叶酸对人脐静脉血管内皮细胞(HUVEC)组织型纤溶酶原激活物(tPA)含量及其mRNA表达的影响. 方法将体外培养的HUVEC分为10个实验组0、10、50、200、500μmol/L 浓度Hcy组及叶酸(15μmol/L)和上述各Hcy共同培养组,培养24*!h后,酶联免疫吸附实验法(ELISA)测定各组细胞上清液中的tPA含量,反转录聚合酶链反应(RT-PCR)半定量分析各组tPA mRNA表达水平. 结果与单纯培养基组(0μmol/L Hcy)相比,10μmol/L Hcy组(生理浓度组)tPA 含量及mRNA表达明显增高(P<0.05).超生理剂量Hcy时,tPA含量及mRNA表达剂量依赖性地下降,但与对照组差异无显著性(P>0.05).而与生理浓度Hcy相比,当Hcy浓度达到500μmol/L时,tPA合成及mRNA表达水平均明显减少(P<0.05).加入叶酸后,可以减弱Hcy抑制tPA合成及mRNA表达的作用,500μmol/L Hcy共同培养组与单纯Hcy组相比具有统计学意义(P<0.05). 结论高Hcy可下调tPA 的mRNA表达,减少内皮细胞tPA的分泌,可能降低纤溶系统的活性.叶酸则可减少高Hcy引起内皮细胞纤溶系统的损害,起到保护作用.生理浓度的Hcy可上调tPA 的mRNA表达,增加内皮细胞tPA的分泌,可能提高纤溶系统的活性.     

人脐静脉内皮细胞分泌组织型纤溶酶原激活物及其抑制剂1对醒脑静干预的反应

背景：前期研究发现醒脑静能有效抑制兔心肌缺血再灌注模型的炎症递质及促纤溶作用,但是影响纤溶系统活性的作用机制未完全明确。目的：观察醒脑静对重组人肿瘤坏死因子α介导人脐静脉内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活物抑制剂1及其基因表达的影响。方法：取3-5 代人脐静脉内皮细胞,在培养基中添加10 μg/L 重组人肿瘤坏死因子α介导人脐静脉内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活物抑制剂1及其基因表达,醒脑静组加入不同浓度(5,10,20 mL/L)的醒脑静干预,阳性对照组添加氟伐他汀(1 μmol/L),并设立单纯人脐静脉内皮细胞培养的空白对照组。培育24 h后采用酶联免疫吸附双抗体夹心法(ELISA)检测细胞上清液组织型纤溶酶原激活物和纤溶酶原激活物抑制剂1水平;采用反转录聚合酶链反应检测人脐静脉内皮细胞的组织型纤溶酶原激活物和纤溶酶原激活物抑制剂1的mRNA表达。结果与结论：重组人肿瘤坏死因子α组纤溶酶原激活物抑制剂1分泌和mRNA表达较空白对照组显著升高 (P < 0.05),组织型纤溶酶原激活物分泌和mRNA表达较空白对照组显著降低(P < 0.05)。不同浓度醒脑静组纤溶酶原激活物抑制剂1分泌和mRNA表达均较重组人肿瘤坏死因子α组显著降低(P < 0.05),而组织型纤溶酶原激活物分泌和mRNA表达均较重组人肿瘤坏死因子α组显著升高(P < 0.05),且呈剂量依赖关系。结果证实,醒脑静作用可逆转重组人肿瘤坏死因子α所致的人脐静脉内皮细胞的纤溶活性。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

辛伐他汀对尼古丁诱导脐静脉 内皮细胞分泌t-PA和PAI-1的影响

目的: 研究不同浓度辛伐他汀对尼古丁诱导人脐静脉内皮细胞(HUVECs)分泌组织纤溶酶原激活物(t-PA)和1型纤溶酶原激活物抑制剂(PAI-1)及其基因表达的影响。方法: 将3-6代体外培养的HUVECs随机分为对照组、尼古丁组及不同浓度辛伐他汀组,辛伐他汀组分别以1、10、100 μmol/L辛伐他汀预处理细胞2 h,再以100 μmol/L尼古丁孵育24 h。酶联免疫吸附双抗体夹心法(ELISA)检测细胞上清液t-PA和PAI-1含量;逆转录聚合酶链反应(RT-PCR)检测细胞t-PA和PAI-1 mRNA的表达。结果: 尼古丁组PAI-1分泌和mRNA表达较对照组显著升高(P<0.05)。不同浓度辛伐他汀组PAI-1分泌和mRNA表达均较尼古丁组显著降低,且PAI-1分泌和mRNA表达的降低呈浓度依赖性(均P<0.05),以100 μmol/L辛伐他汀组最为显著。100 μmol/L辛伐他汀组PAI-1分泌和mRNA表达与对照组比较,无显著差异(P>0.05)。尼古丁组t-PA mRNA表达较对照组显著降低(P<0.05)。10、100 μmol/L辛伐他汀组t-PA mRNA表达较尼古丁组显著升高(P<0.05),各组间t-PA分泌无显著差异(均P>0.05)。结论: 在体外,辛伐他汀可降低尼古丁所致的PAI-1分泌和mRNA的表达,并升高t-PA mRNA的表达,从而逆转尼古丁介导的HUVECs纤溶活性减低。     

人参皂甙对内毒素致血管内皮细胞表达TF和PAI-1的影响

目的:研究人参皂甙对内毒素脂多糖(LPS)致血管内皮细胞组织因子(TF)和纤溶酶原激活物抑制剂1(PAI-1)表达的影响,探讨人参皂甙对心血管疾病的保健及防治机制。方法:用酶消化法培养人脐静脉内皮细胞(HUVEC);ELISA方法检测HUVEC条件培养液PAI-1蛋白量;用一步凝固法检测HUVECTF活性;Northernblot检测HUVECTF和PAI-1的mRNA表达。结果:LPS能使HUVECPAI-1蛋白和TF活性及其mRNA表达显著增强,人参皂甙则明显抑制LPS对HUVEC的这种作用。结论:通过拮抗LPS致HUVECTF和PAI-1的表达,人参皂甙可维持血管内皮功能稳定,而发挥其心血管疾病的保健与防治作用。     

氧化低密度脂蛋白内皮受体在氧化低密度脂蛋白致内皮细胞损伤中的作用

目的:探讨血凝素样氧化低密度脂蛋白受体(LOX1)在氧化低密度脂蛋白(ox-LDL)致内皮细胞损伤中的作用。方法:采用倒置相差显微镜观察细胞形态的改变;发色底物法检测内皮细胞培养液中的组织纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制剂(PAI-1)的活性;反转录聚合酶链反应(RT-PCR)检测LOX1mRNA表达水平。结果:单纯加入ox-LDL,内皮细胞出现胞体收缩,细胞膜破坏等明显的损伤性改变,培养液中PAI-1活性较对照组高3倍(P<0.05),LOX1mRNA水平表达增高;而当培养液中同时加有LOX1抑制剂polyinosinicacid时,内皮细胞形态损伤不明显,培养液中PAI-1活性低约2.6倍(P<0.05),同时LOX1mRNA水平降低。结论:LOX1介导了ox-LDL对内皮细胞的损伤,可能在血管损伤性疾病的发生中起重要作用。     

Tissue plasminogen activator induced by dengue virus infection of human endothelial cells

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of dengue virus (DV) infection. However, the pathogenesis of hemorrhage induced by dengue virus infection is poorly understood. Since endothelial cells play a pivotal role in the regulation of hemostasis, we studied the effect of DV infection on the production of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in vitro using both primary isolated endothelial cells, human umbilical cord veins cells, and a human microvascular endothelial cell line. DV infection significantly induced the secretion of tPA but not PAI-1 of human endothelial cells. In addition, tPA mRNA of endothelial cells was induced by DV as demonstrated by RT-PCR. Antibody against IL-6 but not control antibody inhibited DV-induced tPA production of endothelial cells. Furthermore, a good correlation between sera levels of IL-6 and tPA was found in DHF but not DF patients. These results suggest that IL-6 can regulate DV-induced tPA production of endothelial cells, which may play important roles in the pathogenic development of DHF/DSS.     

Endocrinology and Paracrinology: Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured monkey Sertoli cells

Sertoli cells play a central role in the control and maintenanceof spermatogenesis. Isolated Sertoli cells of mouse and rattestes have been shown to secrete plasminogen activator (PA)and a plasminogen activator inhibitor type-1 (PAI-1) in culture.In this study, we have investigated the hormonal regulationof PA and PAI-1 activities in cultured monkey Sertoli cells.Sertoli cells (5x105 cells/well) isolated from infant rhesusmonkey testes were preincubated at 35°C for 16 h in 24-wellplates precoated with poly(D-lysine) (5 µg/cm²) in 0.5ml McCoy's 5a medium containing 5% of fetal calf serum and furtherincubated for 48 h in 0.5 ml serum-free medium with or withoutvarious hormones or other compounds. PA as well as PAI-1 activitiesin the conditioned media were assayed by fibrin overlay andreverse fibrin autography techniques respectively. The Sertolicells in vitro secreted only tissue-type PA (tPA), no detectableamount of urokinase-type PA (uPA) could be observed. MonkeySertoli cells were also capable of secreting PAI-1. Immunocytochemicalstudies indicated that both tPA and PAI-1 positive staininglocalized in the Sertoli cells, spermatids and residual bodiesof the seminiferous epithelium; Northern blot analysis furtherconfirmed the presence of both tPA and PAI-1 mRNA in monkeySertoli cells. Addition of follicle-stimulating hormone (FSH)or cyclic adenosine monophosphate (cAMP) derivatives or cAMP-generatingagents and gonadotrophin-releasing hormone (GnRH) agonist orphorbol ester (PMA) to the cell culture significantly increasedtPA activity. PAI-1 activity in the culture was also enhancedby these reagents except 8-bromo-dibutyryl-cAMP, forskolin and3-isobutyl-1-methylxanthin (MIX) which greatly stimulated tPAactivity, whereas decreased PAI-1 activity, implying that neutralizationof PAI-1 activity by the high level of tPA in the conditionedmedia may occur. These data suggest that increased intracellularsignals which activate protein kinase A (PKA), or protein kinaseC (PKC) can modulate Sertoli cell tPA and PAI-1 activities.The concomitant induction of PA and PAI-1 by the same reagentsin the Sertoli cells may reflect a finely tuned regulatory mechanismin which PAI-1 could limit the excession of the proteolysis.     

组织型纤溶酶原激活因子及其抑制因子在孕鼠子宫中的表达

本实验用原位杂交和免疫组织化学定位方法从ｍＲＮＡ和蛋白质水平研究组织型纤维溶酶原激活因子（ｔＰＡ）及其Ｉ型抑制因子（ＰＡＩ－１）在受孕第七天大鼠子宫中的表达，结果表明，在非着床点子宫腔上皮细胞，腔上皮附近的基质细胞和腺上皮细胞中ｔＰＡ和ＰＡＩ－ｍＲＮＡ都有表达，但ＰＡＩ－１ｍＲＮＡ的量很低，ｔＰＡ抗原分布于了宫腔上皮和腺上皮，ＰＡＩ－１抗原只能在腺上皮和表层基质中微弱地检测到，在着床点，ｔＰＡ和Ｐ     

Potentiating effect of endothelial cells on astrocytic plasminogen activator inhibitor type-1 gene expression in an in vitro model of the blood–brain barrier

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9–11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.     

mmLDL对人脐静脉内皮细胞PAI-1表达的影响及其转录调控机制

目的:探讨弱氧化修饰低密度脂蛋白(mmLDL)对人脐静脉内皮细胞(HUVEC)PAI-1活性和mRNA表达的影响及其转录调控机制。方法:人脐静脉内皮细胞的培养和鉴定。用发色底物法测定PAI-1活性。Northern印迹分析法检测PAI-1mRNA的水平。采用基因重组技术构建含不同长度PAI-1 5'上游序列的荧光素酶报告基因质粒, 瞬时转染进入内皮细胞, 并检测荧光素酶的表达情况。 利用PCR和测序技术, 对构建质粒上AP-1元件进行定点突变。Western blot印迹杂交检测内皮细胞核内激活蛋白-1(AP-1)蛋白水平。结果:50 mg/L mmLDL诱导HUVECs PAI-1活性和mRNA表达量明显增高, 同时提高核内AP-1蛋白水平。mmLDL显著诱导构建质粒pGL3-PAI-1-1509/+90和pGL3-PAI-1-823/+90的荧光素酶活性, 但对质粒pGL3-PAI-1-553/+90和pGL3-PAI-1-47/+90诱导作用不明显。当PAI-1 5'上游序列的3个AP-1元件突变后, mmLDL的诱导作用明显降低。结论:(1)mmLDL增强血管内皮细胞 PAI-1活性与mRNA表达;(2)PAI-1活性提高与其mRNA表达增加呈正相关;(3) PAI－1 5'上游序列中3个AP-1元件在mmLDL对PAI－1诱导中具有重要调控作用。     

Increased release of tissue plasminogen activator and its inhibitor in human parotid saliva upon stimulation

Parotid saliva from 12 healthy volunteers was collected prior to and after 5 and 25 min of stimulation at a constant flow rate of 0.25 or 1.0 ml min^-1. In the salivary samples the concentrations of tPA (tissue-type plasminogen activator), PAI-1 (plasminogen activator inhibitor type-1), albumin and total protein were determined and the activity of amylase, tPA and PAI assessed. Presence of both tPA and PAI-1 antigen was demonstrated in all samples, and in unstimulated saliva the ratio between the activator and its inhibitor was 1:7. Upon stimulation we found a significantly increased concentration of PAI-1, a less pronounced increase in tPA concentration, unchanged amylase and total protein levels and significantly decreased albumin concentration. tPA activity was significantly reduced after prolonged stimulation which had no effect on PAI activity. In stimulated saliva a significant positive correlation between concentration of tPA and PAI-1 was demonstrated. Stimulation with citric acid had no effect on output of albumin which is passively filtered from blood, whereas the increase in flow rate corresponded to the significantly increased secretion rate of total protein and amylase which is secreted by gland cells. The secretion pattern of tPA and PAI-1 differed significantly from that of albumin in showing markedly increased output rate during the stimulation period, and the relative increase in output of PAI-1 was significantly higher than that of amylase and total protein. Thus, the results from this study suggest an active release of both tPA and its main inhibitor PAI-1 into saliva.