组织纤溶酶原激活剂及其抑制物的研究进展

纤溶酶原转变成有纤溶活性的纤溶酶是纤溶过程中决定性的一步。组织型纤溶酶原激活剂（ｔｉｓｓｕｅｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｔＰＡ）是体内主要的纤溶酶原激活物,而纤溶酶原激活剂抑制物（ｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒｉｎｈｉｂｉｔｏｒ,ＰＡＩ）是组织型纤溶酶原激活剂（ｔＰＡ）的快速抑制剂。近年来国内大量的研究发现,ｔＰＡ、ＰＡＩ活性或含量的变化与许多疾病,特别是心、脑血管疾病密切相关,已引起专家学者日益广泛的重视［１,２］。本文就组织纤溶酶原激活剂及其抑制剂的分子…     

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。     

人组织型纤溶酶原激活剂及其生理性抑制剂的结构与功能

一、人组织型纤溶酶原激活剂的结构和功能 人组织型纤溶酶原激活剂(Tissueplasminogen Activator,t-PA)由527个氨基酸组成,分子量约为67000,属丝氨酸类蛋白水解酶。它能使纤溶酶原(Plasminogen,Plg)变成纤溶酶(plasmin,plm),后者促使水不溶性纤维蛋白(Fibrin,Fb)水     

抗单链尿激酶型纤溶酶原激活剂单克隆抗体的制备

尿激酶型纤溶酶原激活剂（ｕＰＡ）是近年来研究较多的溶血栓药物之一。它存在两种形式：单链尿激酶（ｓｃｕＰＡ）或称尿激酶原及双链尿激酶（ｕＰＡ）或称尿激酶〔１〕。由于临床发现ｕＰＡ大剂量注射易出现大面积出血的倾向，所以，人们对特异性强于ｕＰＡ的ｓｃｕＰＡ进行了深入地研究。由于ｓｃｕＰＡ能选择性地在纤维蛋白表面激活纤溶酶原，进而选择性地溶解血栓，而受到人们的重视。目前，我们虽已构建了高表达水平的工程细胞株，但纯化工艺有待进一步完善。鉴于国内尚未报道过ｓｃｕＰＡ单克隆抗体（ｍＡｂ）的制备，我们用常规细…     

纤溶酶原激活剂抑制物2型抑制细胞凋亡的研究进展

纤溶酶原激活剂抑制物2型(PAI-2)是一种多功能蛋白质,除了能有效抑制尿激酶(uPA)和双链组织型纤溶酶原激活物(tPA)而调节纤溶活性外,还参与了很多其它的生理病理过程,例如组织重建、胚胎发育、感染、免疫系统发育、肿瘤浸润和迁移等。更有研究表明胞内型PAI-2在抑制细胞凋亡方面也发挥着重要作用。本文就近年来PAI-2抑制细胞凋亡的研究进展作一综述。     

胃癌组织中尿激酶型纤溶酶原激活剂及其受体基因表达和临床意义

目的：研究尿激酶型纤溶酶原激活剂（ｕＰＡ）及其特异受体（ｕＰＡＲ）与胃癌的关系及其在胃癌浸润转移中的作用。方法：采用ｃＤＮＡ－ｍＲＡＮ原位分子杂交技术,分别检测了６４例胃癌及其癌旁组织中ｕＰＡ和ｕＰＡＲｍＲＮＡ表达情况,同时结合病人的临床生物学指征进行分析。结果：癌与癌周比较,ｕＰＡ和ｕＰＡＲｍＲＮＡ阳性表达率明显升高,Ｐ〈０．００１。在伴有淋巴结转移的病例中,ｕＰＡ和ｕＰＡＲ阳性例数分别为１９／２９和２４／２９,与无转移的１０／３５和１３／３５相比,Ｐ分别〈０．０１和〈０．００１。在浸润到肌层、浆膜层的病例中ｕＰＡ和ｕＰＡＲ阳性例数分别为２５／３８和３０／３８,与浸润到粘膜和粘膜下层的４／２６例和７／２６相比,Ｐ均〈０．００１。在ｕＰＡ和ｕＰＡＲ同时阳性的病例中,伴有淋巴结转移和浸润到肌层以下的分别占１５／２     

抗PAI抑制作用的组织纤溶酶原激活剂在大肠杆菌中的表达

目的:构建t-PA活性不被PAI-1抑制的新一代t-PA突变体。方法:根据t-PA的结构特点,去除t-PA分子中的指状区、表皮生长因子和Kringle1区,以含全长t-PA编码区序列的pUC18质粒为模板,经PCR扩增编码氨基酸1~3和176~527位的截短式t-PADNA序列;并将该t-PA分子中的PAI-1结合位点,即第373~384位核苷酸(AAGCACAGGAGG)突变为(GCGGCCGCGGCG),相应的氨基酸KHRR则变为AAAA。结果:测序证实,t-PA突变体的DNA序列正确,将其克隆于大肠杆菌表达载体中,并在大肠杆菌中得到高效表达。表达蛋白占总菌体蛋白的30%,以包涵体形式存在。经蛋白质变性、复性,得到有活性的t-PA突变体。t-PA突变体与PAI-1反应后t-PA的活性未受到抑制。结论:t-PA突变体可能是一种用于治疗心肌梗死和脑血栓等血栓性疾病的强效且剂量要求低的新型生物工程药物。     

含人组织型纤溶酶原激活剂cDNA重组逆转录病毒的制备及鉴定

将人组织型纤溶酶原激活剂ｃＤＮＡ与逆转录病毒载体ＬＮＳＸ重组后转染病毒包装细胞ＰＡ３１７，形成完整的重组病毒颗粒，电镜下重组病毒呈散在分布，球形，直径９０～１８０ｎｍ，由囊膜、外壳和核心三部分组成。重组病毒颗粒感染ＮＩＨ３Ｔ３细胞，在含Ｇ４１８培养基中筛选培养２周后计数阳性细胞克隆数，结果达６×１０８ＣＦＵ／Ｌ；被感染的受体细胞ＮＩＨ３Ｔ３高效表达具有纤溶活性的重组人组织型纤溶酶原激活剂。     

颞下颌关节盘前移位对髁状突软骨中纤溶酶原激活剂系统表达的影响

目的研究颞下颌关节盘前移位对关节软骨组织中尿激酶型纤溶酶原激活剂（uPA）系统的影响.方法24只日本大耳白兔在不打开关节囊的情况下建立右侧颞下颌关节盘前移位动物模型,分别于术后4、1、2、4、8d和12周各处死4只动物.常规HE染色观察髁突软骨的形态学变化,并以原位杂交法检测髁突软骨细胞中uPA和纤溶酶原激活剂抑制剂-1（PAI-1）的基因表达变化.结果术后髁突受力区出现一过性的软骨变薄、各层排列紊乱等病理变化.uPA和PAI-1基因转录水平在术后即开始上调,至2周时达到最高水平,至12周时基本恢复至正常水平,与髁突软骨的适应性改建相一致.结论颞下颌关节盘前移位后,关节软骨内uPA系统的变化与关节软骨的适应性改建密切相关.     

纤溶酶原激活系统在肺癌中的表达和意义

正常组织及肿瘤组织的破坏和重建依赖细胞外基质的蛋白水解作用。在多种不同的蛋白酶中，一些丝氨酸蛋白酶（如纤溶酶原激活剂及其激活产物）具有调节肿瘤生长和播散的功能，与多种恶性肿瘤的恶性度及其侵袭和转移性密切相关。作者就纤溶酶原激活系统各主要成分的结构、功...     

TISSUE PLASMINOGEN ACTIVATOR PRODUCTION BY MONOCYTES IN VENOUS THROMBOLYSIS

Laminated occlusive thrombus was induced in the rat inferior vena cava (IVC) by a distal stenosis and injection of thrombin. Immunocytochemistry was performed on serial cryostat sections of the thrombus for tissue plasminogen activator (tPA) and a variety of phenotype markers for mononuclear cells. There was little tPA in 2-day-old thrombus. However, tPA was present in significant quantities in 1- and 2-week-old thrombus. Most of the staining for tPA was associated with monocytes, which had infiltrated the thrombus in large numbers. No caval endothelium was seen in these sections. By 4 weeks, the IVC had re-canalized and new endothelium had formed; tPA staining was weakly positive in the endothelium and smooth muscle. In situ hybridization with a digoxigenin-labelled RNA probe confirmed the monocytes as the main source of tPA. This study shows that large numbers of infiltrating monocytes are present in venous thrombosis and that they are the main source of tPA.     

缺氧对血脑屏障细胞分泌组织型纤溶酶原激活物的影响

目的：探讨缺氧对体外培养鼠脑微血管内皮细胞和星形胶质细胞分泌组织型纤溶酶原激活物（ｔｉｓｓｕｅ－ｔｙｐｅ ｐｌａｓｍｉｎｏｇｅｎ ａｃｔｉｖａｔｏｒ,ＴＰＡ）的影响。方法：分别对新生小鼠脑微血管内皮细胞、星状胶质细胞进行缺氧条件下培养,常规培养作为空白对照,每组各取８例,吸取培养液用酶联免疫吸附试验测试ＴＰＡ活性。结果：缺氧后内皮细胞ＴＰＡ活性明显增高（Ｐ＜０．０１）,星形胶质细胞ＴＰＡ活性无明显变化（Ｐ＞０．０５）。结论：体外培养鼠脑微血管内皮细胞和星形胶质细胞均能合成分泌ＴＰＡ;缺氧状态下脑微血管内皮细胞产生的ＴＰＡ活性增高。内皮细胞分泌的ＴＰＡ是脑缺氧缺血致不可逆神经元损伤的一个重要媒介,而星形胶质细胞分泌的ＴＰＡ则主要参与发育阶段需要细胞迁移的重建活动。     

新生鼠缺氧缺血时脑TPA活性与微血管基膜的相关性研究

为了探讨缺氧缺血时脑内组织型纤溶酶原激活物 (TPA)与脑微血管基膜降解的相关性 ,本研究采用了下述二种方法 :第一组是将一日龄 SD大鼠分为五组 :(1)空白对照组 ,(2 )假手术组 ,(3 )缺氧缺血组 ,(4 )缺氧缺血后复氧 2 4h组 ,(5 )缺氧缺血后复氧 48h组 ,每组 12只。每组取 4例测 TPA活性和 8例鼠脑用抗 型胶原、层粘连蛋白和纤维粘连蛋白抗体标记。第二组是脑微血管内皮细胞和星形胶质细胞体外培养 :分为 (1)空白对照组 ,在常规条件下培养的细胞 ;(2 )缺氧组 ,在培养液表面覆盖无菌医用液体石蜡 ,形成缺氧环境 ,每组取 8例培养液测 TPA活性。结果证明 ,在三个实验组中以缺氧缺血组的 TPA活性最高 ,而后随着复氧时间的增加而下降。培养的内皮细胞缺氧组 TPA活性比对照组高 ,而星形胶质细胞缺氧组 TPA活性与对照组无差别。三个实验组的 型胶原、层粘连蛋白和纤维粘连蛋白阳性染色平均单位面积较两对照组者小。三个实验组阳性产物呈不连续线状的微血管数较两对照组多。以上结果显示 ,缺氧缺血可激发新生大鼠脑内 TPA活性增高 ,主要是脑微血管内皮分泌的 TPA活性增高 ,然后通过一系列酶促反应 ,使微血管基膜的细胞外基质成分— 型胶原、层粘连蛋白和纤维粘连蛋白等降解 ,血脑屏障受损 ,微血管的渗透性增?     

围产期缺氧缺血性脑损伤时基膜与TPA变化的相关性

为了探讨缺氧缺血后基膜与脑组织纤溶酶原激活剂变化的相关性。本实验通过“延迟剖宫产术”致胎鼠宫内窘迫 ;实验分空白对照组、缺氧缺血 15 min组和缺氧缺血 3 0 min组。上述三组分别取额、顶叶 ,透射电镜下观察血脑屏障的变化 ,每组各取8例测试脑组织纤溶酶原激活剂的活性。结果显示 :缺氧缺血 15 min时部分星形胶质细胞足板肿胀、血管周隙扩张 ;缺氧缺血 3 0min,基膜样物质减少 ,神经元显著肿胀。缺氧缺血 15 min、3 0 min,组织纤溶酶原激活剂活性升高 ,并且随缺氧缺血时间的延长 ,组织纤溶酶原激活剂呈增高趋势 ,经 t检验 ,P<0 .0 1。以上结果表明 :缺氧缺血后脑组织纤溶酶原激活剂活性增高 ,引起毛细血管基膜降解 ,打开血脑屏障 ,导致脑水肿。组织纤溶酶原激活剂是脑缺氧缺血引起神经元不可逆损伤的一个重要媒介     

The urokinase plasminogen activator (u-PA) and its inhibitor (PAI-1) in embryo-fetal bone formation in the human: an immunohistochemical study

The role of urokinase plasminogen activator and plasminogen activator inhibitor-1 in human embryofetal bone formation between the 9th and the 20th week of gestation has been studied immunohistochemically. While mature osteocytes of the secondary spongiosa and resting chondrocytes of the bone epiphyses were negative for both antigens in each developmental stage, metabolically active parts of the osseocartilaginous system showed a strong immunoreactivity. Until the end of the 10th week of gestation urokinase plasminogen activator and plasminogen activator inhibitor-1 could not be demonstrated in the shaft of the preexisting cartilaginous models of bones, which correlates with the morphological developmental stage of the embryos. Later, osteoblasts and chondrocytes in the areas of enchondral ossification, and the perivascular chondrocytes of the epiphyseal secondary ossification centres, showed similarly high concentrations of urokinase plasminogen activator and plasminogen activator inhibitor-1. Moreover, the individual ossification stages of the different bones in embryo-fetal development could be demonstrated immunohistochemically. While humeri and femora showed diaphyseal immunoreactivities at an early stage, positive reactions in the phalanges were found only much later. Thus, the enzymes of the fibrinolytic system studied are clearly involved in the desmal and enchondral ossification process in the osseocartilaginous compartment.     

Prognostic relevance of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in gastric cancer

Expression of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was evaluated in 125 surgically resected gastric cancers by immunohistochemical analysis. Tissue was stained immunohistochemically with a monoclonal antibody against human uPA and monoclonal antibodies against human PAI-1 and PAI-2. In addition, DNA ploidy patterns were determined by cytofluorometer after staining with propidium iodide. We found that 82 (66%) of the 125 gastric cancers expressed uPA as diffuse cytoplasmic staining, as intensely outlined luminal borders. PAI-1 expression was observed in 62 (50%) of 125 gastric cancer as a fine, diffuse and granular pattern in the cytoplasm. PAI-2 expression was observed in 65 (52%) of the 125 gastric cancers as a diffuse cytoplasmic staining. uPA-positive tumours showed a higher incidence of infiltration, lymph node metastasis and peritoneal dissemination than uPA-negative ones. Patients with uPA-positive tumours proved to have a significantly poorer prognosis than those with negative ones. PAI-1-negative tumours showed a higher incidence of liver metastasis and carried a poorer prognosis than PAI-1-positive ones. There was no significant correlation between uPA or PAI-1 expression and DNA ploidy patterns. Conversely, there was no significant relationship between PAI-2 expression and clinicopathological parameters and prognosis. According to the expression of uPA and PAI-1 status, groups of 19 uPA(–)/PAI-1(–), 44 uPA(+)/PAI-1(–), 23 uPA(–)/PAI-1(+) and 39 uPA(+)/PAI-1(+) were subdivided. Tumours with UPA(+)/PAI-1(–) had a significantly higher incidence of liver metastasis, lymph node metastasis and serosal invasion than the other groups of tumours. Patients with uPA(+)/PAI-1(–) tumours had a significantly poorer prognosis than those with uPA(–)/PAI-1(+) tumours. These results indicate that uPA expression is a useful biological prognostic indicator, and that uPA and PAI-1 may play an important part in the tumour progression and metastasis in gastric cancer.     

高血压病各期纤溶活性变化的意义

进一步探讨高血压病各期纤溶活性变化的临床意义 ,为早期防治寻找依据。方法用发色底物法测定血浆纤溶酶原激活物抑制物(PAI)、组织型纤溶酶原激活物(t-PA)及纤溶酶原(PLG)的含量。结果①高血压病Ⅱ期、急性脑梗塞及脑出血各项指标均与正常组比较有明显差异(P<0.001) ,急性脑出血、脑梗塞较高血压病Ⅱ期t-PA、PAI有明显差异(P<0.01);②脑卒中各组纤溶活性均无明显差异(P>0.05) ;③合并冠心病者与单纯高血压病Ⅱ期比较 ,t -PA、PAI有明显差异(P<0.01)。结论高血压病各期存在纤溶功能的失衡 ,提示早期干预治疗的重要性。测定血浆PAI、t-PA ,有利于判断高血压病的严重程度、疗效、预后     

ANGIOTENSINOGEN AND PLASMINOGEN ACTIVATOR INHIBITOR-1 GENE POLYMORPHISM IN RELATION TO CHRONIC ALLOGRAFT DYSFUNCTION

The role of HLA-antigens in susceptibility to hepatitis C virus (HCV) infection is still being debated. We analyzed HLA phenotype frequencies in two major ethnic groups, namely Egyptian and Saudi nationals. The Egyptian group included 110 patients of whom 55 were HCV positive and the other 55 were HCV negative (control group). The Saudi group included 146 HCV-positive patients and 122 HCV-negative individuals (control group). The results for the Egyptian population revealed increased frequencies of some HLA phenotypes and decreased frequencies of others but without any statistically significant difference. In contrast, in the Saudi population, the HLA-A19 phenotype was significantly increased in the HCV-positive patients when compared with the control group, while significantly decreased frequencies were found for HLA-B8, HLA-DRI and HLA-DR3. Our data suggest that there was no significant association between the HLA phenotypes and the susceptibility to HCV infection among the Egyptian population, while the overall data of the Saudi population seem to indicate that the expression of particular HLA-alleles could be associated with the susceptibility or resistance to the HCV infection. Further studies on larger numbers of patients are needed to support the role of the HLA system in HCV infection. A total of 108 HCV-positive patients underwent renal transplantation at the Jeddah Kidney Center, and the results were compared with 100 age- and sex-matched controls. Graft survival at 36 months was 82% and 86% for the HCV positive and control subjects, respectively, while the patient survival rate was, respectively, 90% and 91%. Our data suggest that the outcome, at least in the short-term, of renal transplantation in HCV-positive patients is very good.     

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma

BackgroundThe urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients.MethodsUPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA.ResultsUPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ² = 8.372, P = 0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P = 0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025).ConclusionsOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.