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。     

低密度脂蛋白受体相关蛋白与纤溶系统

低密度脂蛋白受体相关蛋白(LRP)是近年新发现的一种细胞表面蛋白,为低密度脂蛋白受体(LDLR)家族的一员,能与多种配体结合,参与纤溶系统多种成分的代谢,从而调节纤溶系统功能的稳定。     

我国登革2型／4型病毒株嵌合E基因免疫原性的研究

目的 构建含有我国登革2型／4型病毒株嵌合E基因片段的真核表达质粒,观察重组质粒DNA的免疫原性,为登革多价疫苗的研究提供依据。方法 首先将包含我国登革2型病毒43株E蛋白Ⅰ/Ⅱ抗原区和4型病毒B5株E蛋白Ⅲ抗原区的嵌合E基因片段克隆至真核表达载体pcDNA3.1中,通过测定确定嵌合基因序列的正确性。然后将重组质粒以肌肉注射途径,免疫Balb/C小鼠。通过间接免疫荧光法对采集的鼠血清中的病毒特异抗体进行检测。结果 构建的含有我国登革2型／4型病毒株嵌合E基因的真核表达质粒pcDNA-D2/4,经序列测定表明,导入的嵌合E基因片段的序列是正确的。将重组质粒DNA免疫小鼠,在初次免疫后的第3周,可同时检测到针对登革2型和4型病毒的特异荧光。结论 所构建的含有不同血清型的我国登革病毒株嵌合E基因片段的真核重组质粒,可诱导小鼠同时产生针对两个血清型病毒的特异抗体,该研究为新型登革多价疫苗的研制提供了依据。     

尼古丁对血管内皮细胞释放t-PA及PAI-1的影响

目的: 研究尼古丁对人脐静脉内皮细胞(HUVECs)释放组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)的影响。方法: HUVECs培养后接种于24孔培养板中,随机分为对照组及实验组,分别进行以下实验。(1)以0.1、1、10、100 μmol/L 尼古丁孵育HUVECs,12 h后收集各组上清液;(2)以100 μmol/L尼古丁与HUVECs孵育0、4、6、8、12 及24 h,收集各组上清液。采用ELISA法测定各组t-PA和PAI-1的浓度。结果: HUVECs与不同浓度尼古丁孵育12 h后,100 μmol/L尼古丁组PAI-1蛋白较对照组明显增加(P<0.01);0.1、1及10 μmol/L尼古丁组PAI-1蛋白与对照组比较,均无显著差异(均P>0.05);各浓度组t-PA蛋白与对照组比较,均无显著差异(均P>0.05)。HUVECs 与100 μmol/L的尼古丁分别孵育4 、6 、8 、12 及24 h,各组PAI-1蛋白均较对照组明显升高(P<0.05),且其升高呈时间依赖性;各组t-PA与对照组比较,均无显著差异(均P>0.05)。结论: 尼古丁可抑制HUVECs的纤溶活性,对内皮细胞具有损伤作用。     

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.     

脂肪酸对人血管内皮细胞纤溶酶原激活物抑制剂-1表达的影响

为探讨不同种类脂肪酸对血管内皮细胞(ECs)纤溶酶原激活物抑制剂-1(PAI-1)表达的直接影响及影响水平，以不同浓度的亚麻酸、亚油酸、油酸、硬脂酸诱导培养ECs，逆转录聚合酶链式反应(RT-PCR)和酶联免疫吸附法。（ELISA)检测PAI-1的mRNA和蛋白表达，随后以上述脂肪酸诱导转染了由PAI-1启动子控制表达的报告基因的ECs，ELISA检测PAI-1启动子转录活性，结果ECs中亚麻酸、亚油酸和油酸均浓度依赖地诱导PAI-1 mRNA和蛋白表达，硬脂酸无影响，而它们对PAI-1启动子转录活性的影响与上述作用一致，提示不饱和脂肪酸通过提高PAI-1转录活性诱导其在ECs的表达。     

蚯蚓蛋白激酶对体内纤溶活性的增强作用研究

目的：探讨蚯蚓蛋白激酶制剂（LK）的纤溶增强作用。方法：采用静脉给药方法，观察LK对SD大鼠纤溶激活因子t-PA及其抑制物PAI-1的急性作用。结果：给药后，PAI-1活性被显著抑制（P<0.01），t-PA活性则明显增强（P<0.01）。剂量1200 U/kg体重的LK，其增强t-PA作用显著大于600U/kg体重剂量LK（P<0.05）。此外，体外观察表明:LK具有明显的激活纤溶酶原（Plg）作用，且有量-效关系，具有一定的浓度依赖性（1.56-25 kU/L）。结论：结果表明，LK具备增强纤溶的特性，静脉给药作用快速而肯定。     

纤溶酶原激活剂抑制物2型的结构与功能

纤溶酶原激活剂抑制物2型(PAI-2)具纤溶抑制活性,是尿激酶(uPA)特异的抑制物。u-PA在肿瘤浸润与转移过程中起了十分关键的作用,PAI-2亦因此成为当今研究的热点。本文对PAI-2的基因结构、表达调控、蛋白质结构、功能及其作用机制等进行了综述。     

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。     

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。     

代谢综合征患者纤溶活性与胰岛素抵抗的关系

目的:探讨代谢综合征患者血浆纤溶活性及与胰岛素抵抗的关系。方法:选择单纯代谢综合征患者36例,代谢综合征合并冠心病患者44例和健康人群对照20例。观察一般情况,并测定血浆纤溶酶原激活抑制物-1(PAI-1)和组织型纤溶酶原激活物(t-PA)、纤维蛋白原(Fib)、空腹血糖(FBG)、空腹血胰岛素(Ins)和血脂等指标;计算胰岛素敏感指数(ISI)。结果:代谢综合征患者的ISI较正常对照组明显下降(P<0.05);血浆PAI-1、Fib等明显升高(P<0.05)。代谢综合征合并冠心病组PAI-1与Fib、Ins正相关,与ISI、高密度脂蛋白(HDL)负相关。结论:代谢综合征患者存在明显的胰岛素抵抗和纤溶活性异常,同时纤溶活性异常与胰岛素抵抗相关。     

Changes in the fibrinolytic system associated with physical conditioning

Summary The effects of physical conditioning on plasma fibrinolytic activity were studied in two groups of subjects. Volunteers not engaged in any sport were compared with individuals having been subjected to aerobic conditioning (middle-distance runners, defined as men running more than 80 km per week). Plasma concentrations of the different components of the fibrinolytic system were evaluated before and immediately after a maximal effort treadmill protocol. Comparison of the resting parameters revealed that under basal conditions for plasma concentrations of plasminogen, fibrinogen, 2-antiplasmin, protein C and protein S there were no differences between the two groups. Concentrations of the fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) were significantly higher in the runners than in the control group, indicating an increased fibrinolytic potential that seemed to be a consequence of the reduced formation of tissue plasminogen activator-plasminogen activator inhibitor (t-PA-PAI) complexes. Acute maximal exercise resulted in pronounced fibrinolysis, evidenced by the elevation of FbDP and FgDP concentrations, in both groups of subjects. The acceleration of the fibrinolytic activity was larger in conditioned individuals, which could be accounted for by a higher t-PA release and reduced formation of t-PA-PAI complexes when compared to the untrained subjects.     

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.     

登革病毒感染对血管内皮细胞ICAM-1表达的影响

目的探讨登革病毒2型（DV2）体外感染对人脐静脉内皮细胞（HUVEC）表达细胞间黏附分子（intercellular adhesion molecule，ICAM-1）的影响。方法原代分离培养HUVEC，用生长良好的2、3代细胞进行实验。用CCK-8法测定DV2感染前后的细胞活性。流式细胞仪测定DV2感染组和对照组细胞在不同实验时间点细胞表面ICAM-1蛋白表达的情况。采用逆转录聚合酶链反应（RT-PCR）法和图像定量分析技术分析DV2感染组和对照组在不同实验时间点HUVEC内ICAM-1 mRNA水平。结果病毒感染HUVEC对细胞活力的影响与对照组相比差异无统计学意义。DV2感染HUVEC促进ICAM-1 mRNA转录，DV2组与对照组相比差异有统计学意义（P〈0．05）。其中，正常状态下HUVEC有一定水平ICAM-1 mRNA转录，但感染后显著增加（P〈0．05），24—72h维持于高水平，与其他时间表达差异有统计学意义（P〈0．05）。DV2感染HUVEC，细胞表达ICAM-1蛋白在12～72h显著升高，与对照组相比差异有统计学意义（P〈0．05）。结论DV2感染上调HUVEC的ICAM-1 mRNA水平和蛋白表达，从而诱发并加重血管内皮局部的损伤，破坏血管的屏障功能，促进血管渗漏的形成与发展。这可能是DV2感染诱发患者血管通透性升高和血浆渗漏的重要分子机制之一。     

Urokinase-type plasminogen activator receptor in gastric cancer: tissue expression and prognostic role

The urokinase-type plasminogen activator (UPA) and its inhibitor PAI-1 are thought to play an important part in gastric cancer (GC) invasion and metastasis. Little is known about the behavior and prognostic impact of the receptor for UPA (UPAR). The aims of the present study were: (1) to measure UPAR, UPA and PAI-1 levels in GC and in non-malignant tissue distant from the tumor (NORM); (2) to evaluate their relationship with histomorphological parameters; and (3) to determine their prognostic value. UPAR, UPA and PAI-1 levels were determined by ELISA in GC and NORM samples from 20 patients with GC undergoing surgery. The GC was also examined in terms of the presence (n=10) or absence (n=10) of metastasis, differentiation (five differentiated, 15 undifferentiated) and histotype. Survival was analysed using life table analysis. UPAR, UPA and PAI-1 were significantly higher in GC vs NORM, in the presence of metastasis (UPAR, UPA) and in undifferentiated GC (UPAR, PAI-1). UPAR significantly correlated with UPA and PAI-1. Low levels of UPAR (P=0.04), UPA (P=0.007) and PAI-1 (P=0.02) were associated with a better survival. Our results demonstrate a sharp increase in UPAR in GC and suggest a prognostic role for it. The concomitant activation of UPAR, UPA and PAI-1 in GC confirm the important role of the plasminogen activator system in the process of invasion and metastasis.