尿激酶型纤溶酶原激活物途径在大鼠酒精性肝纤维化形成中的作用

目的 通过观察尿激酶型纤溶酶原激活物(uPA)及其抑制物(PAI-1)在大鼠酒精性肝纤维化形成中的动态变化,探讨uPA纤溶途径在酒精性肝纤维化形成中的作用.方法 雄性SD大鼠随机分为空白组、四氯化碳(CCl4)组和造模组.采用56度二锅头酒、玉米油、吡唑混合物灌胃联合腹腔注射CCl4橄榄油溶液(CCl4∶橄榄油=1∶3...     

尿激酶型纤溶酶原激活物及其抑制物与肝纤维化

肝纤维化是肝脏对慢性损伤的一种修复反应,以细胞外基质(ECM)在肝内过多沉积为特征。尿激酶型纤溶酶原激活物(uPA)及其抑制物(PAI)是调节基质金属蛋白酶(MMP)活性和ECM降解的关键因素。uPA通过uPA-纤溶酶-MMP级联反应途径,最终可产生活化的纤溶酶和MMP,后两者是降解ECM的重要物质。因而调控uPA的表达,可能为肝纤维化的治疗提供新的途径。     

腺病毒介导的人uPA体外转染大鼠骨髓源性肝干细胞的研究

目的评价携带人uPA基因的腺病毒(AduPA)转染骨髓源性肝干细胞(BDLSC)的效率,在BDLSC中的表达及其对BDLSC增殖和分化的影响。方法利用淤胆型肝损伤大鼠模型和含5%淤胆血清病理条件培养液筛选和扩增BDLSC,采用荧光显微镜检测AduPA的转染效率,采用Westernblotting法检测uPA在BDLSC中的表达,采用MTT法和免疫细胞化学法分别检测转染AduPA的BDLSC的增殖和分化变化。结果随着MOI的增高,转染效率明显增高,当MOI为500时,转染效率达92.6±2.2%,转染3天后的人uPA在BDLSC稳定表达,且转染AduPA对BDLSC的增殖和分化无明显影响。结论BDLSC可能是一种良好的基因载体细胞,可携带uPA用于肝纤维化的治疗研究。     

肝硬化患者血浆中尿激酶型纤溶酶激活物的检测及其意义

目的 探讨肝硬化患者血浆尿激酶型纤溶酶激活物(uPA)、尿激酶型纤溶酶激活物受体(uPAR)、纤溶酶原激活物抑制剂-1(PAI-1)的变化及其意义。 方法 确诊的72例乙型肝炎后肝硬化患者,Child-pugh分级A级23例(A组),B级29例(B组),C级20例(C组)。6例健康志愿献血者为正常对照组。酶联免疫吸附实验测定血浆uPA、uPAR、PAI-1的变化。并同时检测血透明质酸(HA)、Ⅳ型胶原(C Ⅳ)、Ⅲ型前胶原(PC Ⅲ)、血浆白蛋白、胆红素、凝血酶原时间及其活动度改变。 结果 随着肝硬化的进展,血浆uPA、uPAR、PAI-1逐渐增加,HA、PC Ⅲ也明显增加。Child C组患者血浆uPA、uPAR、PAI-1水平(μg/L)分别为1.88±0.64、4.82±2.02和52.60±16.87,A组分别为1.36±0.43、3.03±1.48和24.09±7.14,B组分别为1.79±0.62、4.80±2.22和41.40±17.52,C组与A、B组比较,t值为2.81～7.38,P值均<0.01。A组血浆uPA与PC Ⅲ呈负相关(r=-0.4785,P<0.05);C组PAI-1与HA呈正相关(r=0.5447,P<0.01)。 结论 肝硬化晚期,虽然血浆uPA、PAI-1增加,但总的效应表现为uPA相对不足,肝基质纤维降解受抑制,血浆uPA、PAI-1与肝硬化发展密切相关。     

Relationship between plasminogen activator inhibitor‐1 gene alterations and fibrosis in peritoneal dialysis patients

Peritoneal fibrosis (PF) is a pathological change that occurs mostly long‐term peritoneal dialysis (PD) patients, as a result of triggering the inflammatory response. Plasminogen activator inhibitor‐1 (PAI‐1) is an important molecule featured in the development of fibrosis. It has been shown in literature that PAI‐1 gene alterations are associated with fibrosis in many tissues and organs. However, PAI‐1 gene alterations in long‐term PD patients have not yet been investigated. In this study, PAI‐1 4G/5G polymorphism was examined by reverse hybridization, and all coding exons of the PAI‐1 gene were examined by sequence analysis to provide treatment modification in patients with predisposition before fibrosis develops. The patients were divided into two groups according to ultrafiltration failure test and duration of PD treatment: those with suspected PF or a high probability of developing PF (36%) and those with a low probability of developing PF (64%). There was no significant difference between the two groups in findings such as peritoneal equilibration test (PET), Kt/V, the content of the PD solution used, peritonitis, and PAI‐1 4G/5G polymorphism (P > .05). A total of eight gene alterations (rs2227660, rs2227668, rs2854233, rs41281004, rs61553169, rs368413856, rs2227684) were detected by sequence analysis, one of which was exonic (rs6092). When the genotype distributions of these variants were examined, no significant difference was found between the two groups. PAI‐1 gene changes were not detected in patients with the probability of developing PF. There is a need for further studies involving other molecules responsible for predisposing to PF with larger patient populations in patients undergoing long‐term PD treatment.     

实验性肝纤维化大鼠肝组织中纤溶酶原激活物抑制剂-1分布与表达

为动态观察肝纤维化大鼠模型肝组织中PAI-1的分布和表达,采用CC l4制备肝纤维化大鼠模型进行观察试验。通过对模型肝组织进行PAI-1免疫组化染色和采用图文分析系统检测PAI-1面密度。结果如下:PAI-1阳性染色主要分布在肝纤维化活跃的汇管区、肝组织变性坏死处;PAI-1面密度随注射CC l4时间的延长而递增(3周0.1356±1.052E-2;6周0.1979±1.191E-2;9周0.2321±2.159E-2,P<0.05),与α-SMA阳性细胞数、胶原面积、HPY含量变化一致;停止注射四氯化碳3周时PAI-1面密度明显下降(7.966E-2±5.587E-3)低于6周时(P<0.05),与α-SMA阳性细胞数变化一致,而胶原面积、HPY含量与9周时相同,无明显下降。肝组织中PAI-1的表达与星状细胞活化有关,可反映肝纤维化的进展,但不能反映肝纤维化程度。     

Overexpression of hepatic plasminogen activator inhibitor type 1 mRNA in rabbits with fatty liver

INTRODUCTION Plasminogen activator inhibitor type 1 ( PAI-I ), an approximately Mr 50000 glycoprotein, is the major physiological inhibitor of plasminogen activators. It is not only the priming factor for atherosclerosis and coronary thrombosis[1-3] , but also participates in the genesis of chronic hepatitis and liver fibrosis[4-11] . However, there has been no available report yet about the research of hepatic PAl-1 gene expression in hyperlipidemia and fatty liver. The present study aimed to explore the change of hepatic PAl-1 mRNA and its plasma activity by means of animal model.     

Plasma kallikrein clearance by the liver of chronic carbon tetrachloride-treated rats

Abstract We have previously reported that the endocytosis of rat plasma kallikrein (RPK) by hepatocytes is a calcium-independent and beta-galactoside-dependent mechanism. We now report the clearance of RPK by the liver of four groups of rats: normal, inflamed (48 h ex-turpentine) and two groups chronically treated with CCl₄ (52 mg/kg per week, intragastrically, for 9-12 weeks). Each liver was isolated, exsanguinated and perfused at 37°C with 30 mL of BSA-Krebs-Henseleit-bicarbonate medium containing 10 nmol/L RPK. Although all rats received the same mild CCl₄ treatment, the liver histology showed that they evolved either to severe hepatitis (serum alanine aminotransferase [ALT] 4852 ± 885 U/L, parenchymatous necrosis in the perivenous region) or to compensated cirrhosis (serum ALT 209 ± 42 U/L, vigorous fibrous encircling regeneration nodules); neither jaundice nor ascites was noted. The results show that serum albumin was not altered among the groups and that: the acute-phase response by itself (inflamed group) increased RPK clearance rate (3.01 ± 0.59 mL/min) as compared with the normal group (1.85 ± 0.14 mL/min); the CCl₄ treatment, although induced an acute-phase response, decreased ( P < 0.01) RPK clearance rates (0.80 ± 0.11 mL/min hepatitis group and 0.98 ± 0.10 mL/min cirrhosis group). These findings suggest that the hepatic clearance rate of plasma kallikrein is an early indicator of liver injury.     

Urokinase type plasminogen activator receptor expression in colorectal neoplasms

Background—The urokinase type plasminogenactivator receptor (uPAR) may play a critical role in cancer invasionand metastasis.
Aims—To study the involvement of uPAR incolorectal carcinogenesis.
Methods—The cellular expression and localisationof uPAR were investigated in colorectal adenomas and invasivecarcinomas by in situ hybridisation, immunohistochemistry, and northernand western blot analyses.
Results—uPAR mRNA expression was found mainly inthe cytoplasm of dysplastic epithelial cells of 30% of adenomas withmild (19%), moderate (21%), and severe (47%) dysplasia, and in that of carcinomatous cells of 85% of invasive carcinomas: Dukes' stages A(72%), B (93%), and C (91%). Some stromal cells in the adjacent neoplastic epithelium were faintly positive. Immunoreactivity for uPARwas detected in dysplastic epithelial cells of 14% of adenomas and incarcinomatous cells of 49% of invasive carcinomas. uPAR mRNA andprotein concentrations were significantly higher in severe than in mildor moderate dysplasia (p<0.05); they were notably higher in Dukes'stage A than in severe dysplasia (p<0.05), and significantly higher inDukes' stage B than in stage A (p<0.05), but those in stage B werenot different from those in stage C or in metastatic colorectalcarcinomas of the liver.
Conclusions—Colorectal adenoma uPAR, expressedessentially in dysplastic epithelial cells, was upregulated withincreasing severity of atypia, and increased notably during thecritical transition from severe dysplasic adenoma to invasivecarcinoma. These findings implicate uPAR expression in the invasive andmetastatic processes of colorectal cancer.

Keywords:urokinase type plasminogen activator receptor; colorectal adenoma; colorectal cancer; adenoma-carcinoma sequence

     

Treatment of experimental hepatic fibrosis by combinational delivery of urokinase-type plasminogen activator and hepatocyte growth factor genes.

PURPOSE: To investigate the effect of combinational delivery of urokinase-type plasminogen activator (uPA) and hepatocyte growth factor (HGF) genes on hepatic fibrosis. METHODS: Replication-deficient adenoviral vectors expressing either human HGF (AdHGF) or uPA (AduPA) were generated. HGF gene was transferred into primary cultured hepatocytes and uPA gene to hepatic stellate cell (HSC) to investigate the effect on the biological character of cells. Combinational adenoviruses were infused into hepatic fibrosis rats. Serum markers as well as histological and immunohistochemical examination were carried out to test the reversal of hepatic fibrosis. RESULTS: Transfection of exogenous HGF gene induced expression of c-met/HGF receptor and stimulated hepatocyte proliferation. uPA gene delivered into HSC decreased the amount of collagen types I and III accompanied with the increased expression of matrix metalloproteinase-2. In vivo, the area of extracellular matrix in the fibrotic liver decreased to 72% in AdHGF-treated rats (P<0.01), 64% in the AduPA-treated group (P<0.01), and 51% in bi-genes transfection (P<0.01), compared with that of the controls. Moreover, immunohistochemical staining of collagen types I and III revealed that combinational genes delivery exerted more effect on reversal of hepatic fibrosis than mono-gene transfection. CONCLUSIONS: Our study indicated that simultaneous delivery of two antifibrotic genes could confer synergistic effect on hepatic fibrosis.     

Expression of urokinase-type plasminogen activator receptor and plasminogen activator inhibitor-1 in gastric cancer

In gastric cancer, the urokinase-type plasminogen activator (uPA) system plays important roles in invasion and metastasis, processes which entail proteolysis and adhesion. Both the urokinasetype plasminogen activator receptor (uPAR) and the plasminogen activator inhibitor-1 (PAI-1) are thought to be important factors in this system. To clarify the relationship between these two factors and gastric cancer invasiveness, we evaluated the expression of uPAR and PAI-1 in 91 cases of gastric cancer by immunohistochemistry and in situ hybridization. Urokinase-type plasminogen activator receptor-mRNA, PAI-1-mRNA, uPAR and PAI-1 protein were diffusely distributed in the cytoplasm of the cancer cells and concentrated at invasive foci. Urokinase-type plasminogen activator receptor protein expression correlated with lymphatic, venous invasion (P<.01) and lymph node metastasis (P<0.05); uPAR-mRNA expression correlated with lymphatic, venous invasion and lymph node metastasis (P<0.05). Plasminogen activator inhibitor-1 protein expression correlated with lymphatic, venous invasion, lymph node metastasis and depth of invasion (P<0.01); PAI-1-mRNA expression was linked to lymphatic, venous invasion (P<0.01), lymph node metastasis and depth of invasion (P<0.05). This suggests that the proteolytic activity of uPAR and the cellular motility of PAI-1 in gastric cancer cells may determine penetration of lymphatic and blood vessels, whereby lymph node metastasis may be promoted and that the promotion of cellular motility by PAI-1 may influence the depth of cancer invasion.     

Smad7和尿激酶型纤溶酶原激活剂基因共表达对大鼠肝纤维化的治疗作用

目的观察Smad7和尿激酶型纤溶酶原激活剂（uPA）基因共表达对CCl4诱导大鼠肝纤维化的治疗作用。方法40只SD大鼠皮下注射CCl4建立肝纤维化模型。将动物分为模型组、Adsmad7／uPA组（尾静脉注射AdSmad7／uPA）、AdSmad7组（尾静脉注射AdSmad7）和AdGFP组[尾静脉注射仅表达绿色萤光蛋白（GFP）的腺病毒载体]，每组10只，另取10只大鼠作为正常对照组。采用放射免疫法测定血清Ⅲ型前胶原（PCⅢ）和层粘连蛋白（LN）水平；碱水解法检测肝组织羟脯氨酸含量；免疫组化法检测肝组织Smad7和uPA表达；天狼星红染色法检测肝纤维化程度。结果注射3d后，AdSmad7／uPA组肝组织Smad7和uPA表达量均显著增加，血清ALT、AST、PCIl、LN和羟脯氨酸水平较AdGFP和AdSmad7组明显下降（P值均〈0．05）。AdSmad7／uPA组的肝纤维化面积为（5．96±2，72）％，明显低于AdGFP组的（13．32±2．47）％和AdSmad7组的（8．73±2．30）％（P值均〈0．05）。结论Smad7和uPA双基因共表达可协同阻断CCl4诱导大鼠肝纤维化进程。     

高脂血症脂肪肝家兔肝脏纤溶酶原激活物抑制物1型基因表达的研究

目的探讨高脂血症脂肪肝肝组织纤溶酶原激活物抑制１型（ＰＡＩ－１）ｍＲＮＡ的表达以及血浆ＰＡＩ－１活性变化。方法通过持续１２周的高脂饮食建立家兔高脂血症脂肪肝模型,分别利用发色底物法和ＲＴ－ＰＣＲ法测定模型组（ｎ＝１０）血浆ＰＡＩ－１活性及肝脏ＰＡＩ－１ｍＲＮＡ的表达,并设正常饮食组（ｎ＝７）作对照。结果与正常组相比,模型组家兔血浆脂质和ＰＡＩ－１活性在实验６周时即显著增高,实验结束时模型组肝脏呈重     

uPA及V-ATPase在肝细胞癌中的表达及意义

目的研究尿激酶型纤溶酶原激活物（uPA）和ATP动力型质子泵（V-ATPase）在肝癌细胞株的表达,及抑制V-ATPase与癌细胞抑制和凋亡的关系。方法用RT-PCR和Western blot检测稳定转染真核表达质粒pCMV-uPA-HA的HCC97L细胞及转染空载体pCMV-HA的HCC97L细胞中uPA和V-ATPase mRNA和蛋白的表达;在转染uPA的HCC97L细胞培养基中加入不同浓度的Bafilomycin A1,用流式细胞仪和CCK-8分别检测癌细胞凋亡及受抑制情况。结果将uPA转染入低转移潜能肝癌细胞株HCC97L后,uPA mRNA和蛋白表达强于转染空载体的HCC97L,V-ATPase mRNA和蛋白表达随着转染uPA而增强。在细胞培养基中加入Bafilomycin A1后细胞生长增殖受到抑制、凋亡增加,且抑制率和凋亡率均随Bafilomycin A1浓度的加大而增加。结论 V-ATPase mRNA和蛋白表达随癌细胞转移能力的提高而增强;Bafilomycin A1抑制V-ATPase,干扰癌细胞内H＋泵出,从而抑制癌细胞生长增殖、促进癌细胞凋亡。     

基质金属蛋白酶和纤溶酶原激活物及其抑制物在肝纤维化进程中的作用

肝纤维化是动态的病理过程，是反复肝损伤引起细胞外基质（ECM）合成和降解失衡，导致其过度沉积的结果。活化的肝星状细胞（HSC）通过产生细胞外基质蛋白和分泌基质金属蛋白酶（MMPS）在肝纤维化病理生理过程中发挥重要作用。尿激酶型纤溶酶原激活物（uPA）可转化纤溶酶原为纤溶酶，活化MMPs，降解多种ECM成分。纤溶酶原激活物抑制剂-1（PAI-1）具有抑制uPA的作用。基质金属蛋白酶抑制剂1（TIMP-1）抑制MMPs对细胞外基质有降解作用。α平滑肌肌动蛋白（α-SMA）是HSC活化标志。我们测定不同肝纤维化分级中α-SMA、基质金属蛋白-1（MMP-1）、TIMP-1蛋白表达以及血浆uPA、PAI-1的水平，旨在了解它们在肝纤维化发展过程中的作用，从而为临床治疗肝硬化提供理论依据。     

Effects of octreotide on epidermal growth factor receptor,tissue plasminogen activator,and plasminogen activator inhibitor during intraperitoneal adhesion formation

Background . Intraperitoneal adhesions remain a problem after abdominal surgery. Octreotide has been proved to be able to reduce the number, strength, and extent of fibrous bands at and away from the anastomotic site in an animal model of rats with intestinal resection and reanastomosis. The aim of the present study was to investigate whether epidermal growth factor receptor (EGF-R), tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI) are involved in this process. Methods. Laparotomy with intestinal resection and reanastomosis was performed on 60 male Wistar rats. All rats were randomly assigned to five groups: receiving no medication (control; C), normal saline (NS), octreotide solution peritoneal irrigation (Oc), octreotide intramuscular injection (IM), and Oc plus octreotide intramuscular injection (Oc + IM). The concentrations of serum EGF-R, plasma tPA, PAI-1, and PAI-2, and the strength of wound healing were measured. Results. The serum EGF-R concentration showed no significant change from the preoperative level in the C and NS groups 7 and 14 days after the abdominal surgery. However, it decreased significantly on postoperative days 7 and 14 in groups Oc, IM, and Oc + IM (P < 0.05). The plasma tPA concentrations were significantly higher than the preoperative level in all groups of rats on postoperative day 7. The levels were higher in groups Oc, IM, and Oc + IM than in group C or group NS at that time (P < 0.05). On postoperative day 14, the plasma tPA concentrations had returned to the preoperative level in group C and group NS. However, the concentrations in groups Oc, IM, and Oc + IM still remained at a significantly higher level than the concentrations in group C and group NS. The plasma PAI-1 and PAI-2 concentrations showed no significant difference from the preoperative level in group C and group NS on days 7 and 14 after the abdominal surgery. However, the concentrations in groups Oc, IM, and Oc + IM on postoperative days 7 and 14 were markedly lower than those in groups C and NS (P < 0.05). The wound strength was significantly greater on day 14 than on day 7 in all groups. Conclusions. In the rats with octreotide irrigation, the EGF-R level was decreased, the plasma tPA concentration was higher, and the plasma PAI-1 and PAI-2 concentrations were lower when compared with values in group C and group NS rats on days 7 and 14 after surgery. The data suggest that EGF-R, tPA, PAI-1, and PAI-2 are all involved in the mechanism of octreotide's action in reducing adhesion formation. Received: February 25, 2002 / Accepted: November 22, 2002 RID="*" ID="*" Reprint requests to: H.-S. Lai Acknowledgments. This study was supported by a National Science Council Grant (NSC87-2314-B-002-352), and a National Taiwan University Hospital Grant (NTUH. 89S1509)     

Evidence for increased levels of plasminogen activator inhibitor and tissue plasminogen activator in plasma of patients with angiographically verified coronary artery disease

We studied 234 consecutive patients who underwent coronary angiography because of severe angina pectoris. Tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI), and lipoprotein Lp(a) were measured in citrated plasma samples. The 214 patients showing significant coronary artery stenosis (greater than 50% reduction of luminal area in any of the great coronary arteries) had higher mean levels of tPA (P less than 0.001) and PAI (P less than 0.01) than a random population sample of similar age. PAI and tPA levels were higher in smokers than in either non-smokers or ex-smokers, and in patients with hypertension tPA was increased. Subjects with blood group A had a higher mean Lp(a) level than subjects with blood group O. There were positive correlations of PAI and tPA levels with serum triglycerides and with body mass index; Lp(a) correlated weakly with plasma fibrinogen concentrations. The findings suggest an impairment of the fibrinolytic system in patients with coronary artery disease, which offers a link between established risk factors and a plausible pathophysiological mechanism, namely thrombus turnover.     

Abnormal fibrinolysis in healthy male cigarette smokers: role of plasminogen activator inhibitors

The extrinsic fibrinolytic system and its response to cigarette smoking was studied in five healthy male smokers 35-45 years old. Tissue plasminogen activator (t-PA) release in response to venous occlusion was intact both at 8:00 A.M. and 3:00 P.M. Acutely smoking two cigarettes neither stimulated fibrinolysis nor changed levels of t-PA or plasminogen activator inhibitors. Functional plasminogen activator inhibitor (PA-I) levels and euglobulin lysis times were higher in the smoking group than in a control group matched for age, sex, and body mass. Antigenic levels of PA-I 1, the PA-I derived from vascular endothelial cells and platelets, were similar in both groups. While smoking did not acutely alter fibrinolysis in chronic smokers, these individuals had a high frequency of abnormal fibrinolysis characterized by high levels of PA-I activity. This abnormality is due to either high specific activity of PA-I 1 or to the presence of other antigenically distinct plasminogen activator inhibitors. Abnormal fibrinolysis may be one mechanism contributing to the thrombotic diathesis of cigarette smokers.