尖吻蝮蛇毒PCA对血管内皮细胞的影响

<正>目的:观察皖南尖吻蝮蛇毒蛋白C激活剂(PCA)对人脐静脉血管内皮细胞(HUVEC)活性及表达组织因子(TF)、血管性血友病因子(v WF)的影响,探讨PCA对血栓性疾病的防治机制。方法:常规培养HUVEC,MTT法检测细胞活性,ELISA检测细胞上清液中TF和v WF的含量,Western blot法检测细胞内TF蛋白表达,RT-PCR法检测细胞内TF和v WF mRNA的表达。     

人参皂苷Rb1对白消安诱导人脐静脉内皮细胞表达PAI-1、TF和TGF-β1的影响

目的：探讨人参皂苷Rb1对人脐静脉内皮细胞（HUVECs）的保护作用机制。方法:体外培养HUVECs，分别用白消安（BU）和人参皂苷Rb1干预，以RT-PCR、实时荧光定量PCR以及ELISA方法检测其PAI-1、TF和TGF-β₁ mRNA和蛋白的表达。结果:60 mg/L BU使HUVECs PAI-1、TF和TGF-β₁ 蛋白水平明显增高及mRNA表达显著增强，80 mg/L人参皂苷Rb1则可明显抑制BU对HUVECs的这种作用。结论:BU可能通过激活TGF-β₁信号转导途径而上调PAI-1及TF的表达，增强HUVECs促凝活性；人参皂苷Rb1则可能通过抑制这一途径降低PAI-1及TF表达，保护HUVECs，纠正其高凝、低纤溶活性的异常状态。     

人参皂苷Rb1对白消安诱导人脐静脉内皮细胞表达PAI-1、TF和TGF-β1的影响

目的：探讨人参皂苷Rb1对人脐静脉内皮细胞（HUVECs）的保护作用机制。方法：体外培养HU—VECs，分别用白消安（BU）和人参皂苷Rb1干预，以RT—PCR、实时荧光定量PCR以及ELISA方法检测其PAI-1、TF和TGF—β1 mRNA和蛋白的表达。结果：60mg／LBU使HUVECs PAI-1、TF和TGF—β1蛋白水平明显增高及mRNA表达显著增强，80mg／L人参皂苷Rb1则可明显抑制BU对HUVECs的这种作用。结论：BU可能通过激活TGF—β1信号转导途径而上调PAI-1及TF的表达，增强HUVECs促凝活性；人参皂苷Rb1则可能通过抑制这一途径降低PAI-1及TF表达，保护HUVECs，纠正其高凝、低纤溶活性的异常状态。     

人参皂苷Rbl对白消安诱导人脐静脉内皮细胞表达PAl-1、TF和TGF-β1的影响

目的:探讨人参皂苷Rbl对人脐静脉内皮细胞(HUVEC8)的保护作用机制.方法:体外培养HU-VECs,分别用白消安(Bu)和人参皂苷Rbl干预,以RT-PCR、实时荧光定量PCR以及ELISA方法检测其PAl-1、TF和TGF-13,mRNA和蛋白的表达.结果:60 mg/L BU使HUVECs PAl-1、TF和TGF-13I蛋白水平明显增高及mRNA表达显著增强,80 mg/L人参皂苷Rbl则可明显抑制Bu对HUVECs的这种作用.结论:BU可能通过激活TGF-B1信号转导途径而上调PAl-1及TF的表达,增强HUVECs促凝活性;人参皂苷Rbl则可能通过抑制这一途径降低PAl一1及rrF表达,保护HUVECs,纠正其高凝、低纤溶活性的异常状态.     

PTX3对LPS诱导内皮细胞表达TF的影响

目的研究PTX3（pentraxin-3）对脂多糖（LPS）诱导内皮细胞表达组织因子（Tissue factor，TF）的影响及其相关机制。方法胰蛋白酶消化法原代培养人脐静脉内皮细胞（HUVEC），一期凝血法检测不同实验组TF促凝活性，酶联免疫吸附实验测TF抗原和反转录-聚合酶链反应（RT-PCR）测TF mRNA表达。结果与对照组比较，单独加入SAP、CRP和PTX3的M199培养基中HUVEC TF促凝活性、抗原和mRNA表达未见明显改变（P〉0．05）；当与LPS共同培养6h，每组HUVEC TF活性和抗原均明显升高（P〈0．05）。结论PTX3不影响内皮细胞TF促凝活性、抗原和mRNA表达，但参与LPS诱导的内皮细胞表达TF。     

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。     

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。     

脂多糖诱导血管内皮细胞凝血途径相关因子基因表达变化

目的：观察脂多糖(LPS)对脐静脉血管内皮细胞(HUVECs)表达组织因子(TF)、组织因子抑制物(TFPI)和凝血酶调节蛋白(TM)的影响。方法：应用胰酶消化HUVECs并进行传代培养，用生长良好的第2、3代细胞进行实验。同时应用CCK-8测定细胞在不同浓度的LPS(1-100 mg/L)处理前后细胞活性；应用反转录聚合酶链反应(RT-PCR)法检测细胞内TM、TF和TFPI mRNA水平。结果：浓度为10 mg/L的LPS对细胞活力与对照组相比没有显著差异。浓度为10 mg/L的LPS作用使HUVECs显著上调TF mRNA表达，6-24 h可以使细胞TFPI mRNA表达下调,以后渐恢复正常表达，72 h达到正常对照组水平，同时下调TM mRNA表达。结论：LPS(10 mg/L)对HUVECs的活性不造成直接的影响，可显著上调HUVECs的TF mRNA转录，抑制TFPI mRNA 的转录，而不改变TM mRNA的转录，这可能与LPS在感染过程中诱导血栓形成,血液凝固和DIC发生相关。     

PCA通过NF-κB和AP-1信号通路影响HUVECs表达TF

目的:探讨核因子κB(NF-κB)和活化蛋白1(AP-1)信号通路在尖吻蝮蛇毒蛋白C激活剂(PCA)抑制脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVECs)组织因子(TF)表达中的作用。方法:MTT法检测HUVECs活力,免疫组化法检测肿瘤坏死因子受体相关因子6(TRAF6)蛋白在细胞中的分布,Western blot法检测细胞内NF-κB p65、TF、c-Fos和c-Jun蛋白的表达,qPCR法测定TF mRNA在HUVECs的表达,ELISA法检测细胞培养上清中TF的含量。结果:与对照组比较,LPS组的细胞活力明显下降(P0.01),胞质内出现明显的黄染颗粒,胞质染色加深,TRAF6平均吸光度值升高(P0.01),NF-κB p65、c-Jun和c-Fos的蛋白表达均明显增加(P0.01),TF mRNA和蛋白表达亦明显增加(P0.01);PCA+LPS组的细胞活力较LPS组升高(P0.05),细胞形态正常,胞质黄染颗粒不明显,TRAF6平均吸光度明显小于LPS组(P0.01),NF-κB p65、c-Fos和c-Jun 3种蛋白表达则明显降低(P0.01),TF mRNA及蛋白亦表达减少(P0.01)。结论:PCA可明显减轻LPS引起的HUVECs损伤,其机制可能是通过降低TRAF6、NF-κB及AP-1核因子的活化进而减少组织因子的释放来实现的。     

蝮蛇毒血小板抑制因子减轻LPS诱导的内皮细胞损伤

目的:探讨蝮蛇毒血小板抑制因子(AHV-PI)对体外脂多糖(LPS)诱导的人脐静脉血管内皮细胞(HUVECs)损伤的影响及其作用机制。方法:体外培养HUVECs,运用LPS(1 mg/L)诱导HUVECs炎症损伤模型,实验分为空白对照组、LPS组、AHV-PI组和AHV-PI+LPS组。MTT比色法检测HUVECs的活力,倒置显微镜观察HUVECs的形态变化,筛选出AHV-PI最适浓度为5 mg/L。流式细胞术检测细胞凋亡;免疫组化法观察胞内组织型纤溶酶原激活物(T-PA)以及纤溶酶原激活物抑制剂-1(PAI-1)表达情况;ELISA法检测HUVECs上清液中细胞间黏附分子-1(ICAM-1)和组织因子(TF)含量;免疫荧光染色检测胞核内NF-κB亚基p65激活转位情况。结果:LPS组细胞明显梭形化,长宽比增大,呈成纤维细胞状,胞浆出现颗粒样物质。AHV-PI浓度低于5 mg/L时对HUVECs的活性和形态没有明显影响,但可减轻LPS引起的HUVECs的活力抑制和形态改变。与对照组比较,LPS组上清液中TF和ICAM-1含量升高,胞内T-PA和PAI-1表达减少;与LPS组相比,AHV-PI+LPS组上清液中TF和ICAM-1的含量显著降低,细胞内T-PA和PAI-1表达增多,细胞核内NF-κB p65表达减少。结论:AHV-PI能减轻HUVECs损伤,其保护机制与抑制细胞因子分泌及NF-κB活化有关。     

山莨菪碱抗血栓形成的机制研究

观察中药山莨菪碱对内毒素脂多糖所致血管内皮细胞表达纤溶酶原激活物抑制剂１的作用和作用机制。方法用胰蛋白酶消化培养人脐静脉内皮细胞（ＨＵＶＥＸ）;采用酶联免疫吸附试验法和Ｎｏｒｔｈｅｒｎ印迹方法观察ＨＵＶＥＧＣ条件培养液ＰＡＩ－１蛋白量及ｍＲＮＡ表达用免疫细胞学检测ＨＵＶＥＣ核因子ＮＦ－ｋＢ的核内转移情况,结晶显著啬培养ＨＵＶＥＣ的ＰＡＩ－１蛋白和ｍＲＮＡ表达;然而当山莨菪碱和ＬＰＳ同时作用于ＨＵＶ     

登革2型病毒调控血管内皮细胞纤溶系统相关蛋白的表达

目的观察登革2型病毒（DV2）对人脐静脉血管内皮细胞（HUVEC）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。方法应用胰酶消化分离HUVEC并进行传代培养，用生长良好的第2．3代细胞进行试验。用cell counting kit-8（CCK-8）测定DV2感染后细胞活性变化；发色底物法测定感染DV2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DV2感染对细胞活力的影响与对照组相比差异无统计学意义。感染DV2组培养液中tPA活性在12～72h显著升高（P〈0．05）；DV2诱导HUVEC表达tPA mRNA的水平显著上调，12h达到峰值，以后渐降，72h mRNA表达水平仍高于对照组（P〈0．01）。而DV2感染组培养液中PAI-1活性和PAI-1 mRNA的表达与对照组比较差异无统计学意义（P〉0．05）。结论DV2感染可显著上调HUVEC的tPA mRNA转录，增强内皮细胞tPA蛋白的分泌，而不影响PAI-1 mRNA的转录或改变内皮细胞PAI-1的分泌。结果提示DV2可活化但并不损伤内皮细胞，诱发内皮细胞增强表达纤溶酶原激活物而致使纤溶系统失衡，引起纤溶亢进，这可能是诱发DHF／DSS患者急性期出血、低血容量性休克等体征的主要因素之一。     

脑源性神经营养因子对细胞外蛋白水解酶激活作用的研究

 目的：研究脑源性神经营养因子 (BDNF) 对细胞外蛋白水解酶表达和激活作用的影响。 方法：体外分离并培养人脐静脉内皮细胞（HUVEC），RT-PCR法检测HUVEC基质金属蛋白酶MMP-2 、MMP-9 和基质金属蛋白酶组织抑制剂TIMP-1、TIMP-2 mRNA的表达，明胶酶谱检测MMP-2和MMP-9蛋白酶活性，纤维蛋白酶谱检测尿激酶型纤溶酶原激活剂（uPA）蛋白酶活性，Western blotting检测uPA、纤溶酶原激活剂抑制剂（PAI）、TIMP-1及TIMP-2表达。 结果：在对HUVEC增殖无明显促进作用的浓度范围内，BDNF可促进无血清培养的HUVEC MMP-2和MMP-9 mRNA表达,并可促进MMP-2和MMP-9酶原的激活产生活性明胶酶，BDNF对TIMP-1和TIMP-2的表达无明显影响。BDNF以浓度和时间依赖性方式上调HUVEC uPA和PAI-1的表达，并可促进uPA的活性。 结论：BDNF可激活MMPs和uPA/PAI相关的蛋白级联。     

Human cytomegalovirus infection induces mRNA expression and secretion of plasminogen inhibitor type-1 in endothelial cells

The aim of the study was to investigate whether infection of endothelial cells with human cytomegalovirus (HCMV) perturbs expression and production of plasminogen activator inhibitor type-1 (PAI-1). mRNA expression of PAI-1 was investigated by isolating total RNA from HCMV-infected and control cells, followed by Northern blotting and probing with ³²P-labelled PAI-1 probe. Sandwich ELISA was used to investigate PAI-1 production. HCMV induced the expression of PAI-1-mRNA at 2–5 days postinfection (maximum expression was at 3 days postinfection which was 40% higher than control). HCMV also induced secretion of PAI-1 at 2–5 days postinfection. These results indicate that infection of endothelial cells with HCMV disturbs PAI-1 expression and production in these cells. J. Med. Virol. 55:268–271, 1998. © 1998 Wiley-Liss, Inc.     

Possible involvement of toll-like receptor 4 in endothelial cell activation of larger vessels in response to lipopolysaccharide.

Toll-like receptors (TLRs) serve as recognition and signaling elements for bacterial substances. To examine the role of TLRs in endothelial cells of larger vessels in lipopolysaccharide (LPS)-induced signaling, the expression and function of TLRs in human umbilical vein endothelial cells (HUVEC) were analyzed. A high level of TLR4 mRNA expression was found in HUVEC, human peripheral blood mononuclear cells (PBMC) and human monocyte cell line THP-1 cells. Little or no TLR2 mRNA expression was observed in HUVEC. In contrast, strong TLR2 mRNA expression was observed in PBMC and THP-1 cells. Moderate and high levels of TLR1 mRNA expression were found in HUVEC, PBMC and THP-1 cells, respectively. TLR3 mRNA expression was moderate in PBMC but weak in HUVEC and THP-1 cells. Little or no TLR5 and RP105 mRNA expression was observed in HUVEC, whereas a moderate level was detected in PBMC and THP-1 cells. The LPS-induced E-selectin expression in HUVEC was significantly inhibited by pretreatment with an anti-TLR4 mAb. Preincubation of HUVEC with an anti- TLR4 mAb significantly reduced the LPS-induced IL-6 production. LPS induced E-selectin and IL-6 production by HUVEC only in the presence of human serum, suggesting the involvement of soluble CD14. Anti-CD14 mAb strongly inhibited the LPS-induced E-selectin and IL-6 production by HUVEC. The inhibition with the concomitant treatment with anti-TLR4 and anti-CD14 mAbs was stronger than that with anti-CD14 mAb only, although it was slight. These results show that TLR4 in the presence of soluble CD14 plays a major role in the signaling of LPS in endothelial cells of larger vessels.     

Simvastatin inhibits tissue factor and plasminogen activator inhibitor-1 expression of glomerular mesangial cells in hypercholesterolemic rabbits

Tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) activity and/or expression are upregulated in hypercholesterolemia. Despite extensive research on anti-thrombotic effect of statins, little is known about their effects on TF and PAI-1 expression in glomerular mesangial cells under hypercholesterolemic condition. Male rabbits were fed on either normal or high-cholesterol diet for 8 weeks. Then cholesterol-fed rabbits were randomly assigned to simvastatin or starch. At the end of 12 weeks, glomerular mesangial cells were collected. The concentrations of TF and PAI-1 mRNA were detected by RT-PCR. The plasma activities of TF and PAI-1 were determined with enzyme linked immunosorbent assay (ELISA) and chromogenic substrate method, respectively. The atherogenic diet caused a consistent increase in serum concentrations of total cholesterol (TC) and serum triglyceride (TG) (p < 0.05), increased TF and PAI-1 mRNA expression in glomerular mesangial cells and plasma activities as compared to the normal diet (p < 0.01). Four-week simvastatin treatment resulted in significant decrease of mesangial TF and PAI-1 mRNA (p < 0.01), and also of the plasma activities of TF (p < 0.05) and PAI-1 (p < 0.01). These results suggest that simvastatin might protect kidney from the formation of microthrombus under hypercholesterolemic condition and might be a possible pathogenesis of obesity-related glomerulopathy.     

反义转化生长因子β1基因转染大鼠肾系膜细胞及其对纤溶？…

目的 通过对大鼠肾小球系膜细胞（ＭｓＣ）转染反义转化生长因子β１（ＴＧＦ－β１）基因,观察该细胞克隆基质金属蛋白酶家族（ＭＭＰｓ）和纤溶酶原激活物抑制剂１（ＰＡＩ－１）表达的改变,方法 采用脂质体进行基因转染,并用Ｗｅｓｔｅｒｎ ｂｌｏｔ法加以鉴定,后分别应用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ、Ｗｅｓｔｅｒｎ ｂｌｏｔ和酶谱分析法检测该细胞克隆ＭＭＰｓ和ＰＡＩ－１表达的变化。结果 成功地建立低表达ＴＧＦ－     

Reduced monocyte adhesion to aortae of diabetic plasminogen activator inhibitor-1 knockout mice

Objective and design

To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions.

Methods and subjects

Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC).

Treatments

Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL).

Results

The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC.

Conclusion

The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.

     

Procoagulant phenotype of endothelial cells after coculture with biomaterial-treated blood cells

Understanding endothelial cell (EC)/blood/biomaterial interactions is crucial for the advancement of cardiovascular devices that often fail because of the lack of nonthrombogenic biomaterials. To begin to assess these interactions, a static EC/blood cell/biomaterial model was used. Isolated blood cells were pretreated with model biomaterial beads with different surface chemistries: polystyrene (PS), and PS beads grafted with 3-kDa polyethylene glycol (PEG) with either a hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH(2)) terminal group at 5.4 or 54 x 10(4) beads/mL. Biomaterial-treated monocytes, neutrophils, or platelets were applied to human umbilical vein ECs (HUVECs) for 5 or 24 h of static coculture, and the resultant procoagulant HUVEC phenotype was characterized using several methods. Flow cytometry was used to assess surface expression of tissue factor (TF), adenosine triphosphate diphosphohydrolase, phosphatidylserine, and thrombomodulin, a functional TF assay was used to assess TF activity, and a plasma recalcification assay examined clotting times on HUVECs. Static coculture of HUVEC with biomaterial-treated neutrophils induced a procoagulant phenotype as exemplified by upregulation of TF expression and total functional activity, and downregulation of adenosine triphosphate diphosphohydrolase and thrombomodulin expression. The plasma recalcification assay demonstrated that HUVECs cocultured with biomaterial-treated monocytes significantly shortened clotting times, with some effect of similarly treated neutrophils.