Inhibition of high-mobility group box 1 expression by siRNA in rat hepatic stellate cells

AIM:To explore the role of high-mobility group box 1 (HMGB1) protein during liver fibrogenesis and investigate the functional effects of HMGB1 gene silencing in hepatic stellate cells (HSCs) using siRNA.METHODS:Hepatic fibrosis in rats was induced through serial subcutaneous injections of dimethylnitrosamine,and expression of HMGB1 was detected by immunohistochemistry.HMGB1 siRNAs were developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000.HMGB1 expression was evaluated by real-tim...     

转化生长因子β1刺激肝星状细胞中纤溶酶原激活物抑制剂1表达

肝星状细胞(HSC)的激活是肝纤维化发生的中心环节.活化的HSC大量增殖,并合成以胶原为主的细胞外基质(ECM)沉积在肝内.转化生长因子(TGF)β是激活HSC并促进其增殖的最重要细胞因子之一,可促进ECM产生,导致并加速肝纤维化的发生和发展.本实验拟通过免疫细胞化学法检测纤溶酶原激活物抑制剂(PAI)1在HSC中的定位,逆转录聚合酶链反应(RT-PCR)及免疫细胞化学法等研究TGF β1促进PAI 1 mRNA和蛋白质的表达,探讨PAI 1在肝纤维化发生和发展中的作用.     

导入外源人尿激酶型纤溶酶原激活剂基因对肝星状细胞胶原沉积的影响

目的　利用重组复制缺陷型腺病毒载体将外源非分泌型人尿激酶型纤溶酶原激活剂(uPA)基因导入活化的肝星状细胞 (HSC)内 ,研究uPA表达对HSC胶原沉积的影响。方法　细菌内同源重组构建表达非分泌型uPA的复制缺陷型重组腺病毒AduPA。体外感染肝星状细胞株HSC T6 ,Northern印迹法和免疫细胞化学法检测uPA在HSC中的表达。酶联免疫吸附实验测定培养上清基质金属蛋白酶 2 (MMP 2 )分泌水平。免疫细胞荧光法测定外源uPA基因导入对HSC胞质内Ⅰ、Ⅲ型胶原含量变化的影响。结果　同源重组最终获得约 5× 1 0 1 1 efu/mlAduPA。AduPA体外感染HSC 3d后 ,Northern印迹法和免疫细胞化学法显示uPAmRNA及蛋白表达明显增加 ,细胞上清液中MMP 2分泌水平达 (2 74 .4 5± 7.6 3) pg/ml,明显高于对照组的 (1 4 5 .85± 6 .5 8)pg/ml(P <0 .0 1 )。免疫细胞荧光法提示uPA基因导入HSC后 ,胞质内Ⅰ、Ⅲ型胶原含量明显减少。结论　通过重组腺病毒载体将外源uPA基因导入HSC内可维持uPA高效表达并上调MMP 2分泌水平 ,减少细胞外基质沉积 ,是基因治疗肝纤维化的有效方法     

小分子干扰RNA沉默结缔组织生长因子对鼠肝星状细胞培养激活的影响

目的研究化学合成小分子干扰RNA(siRNA)对大鼠原代肝星状细胞(HSC)结缔组织生长因子(CTGF)基因表达的阻抑作用及对HSC培养激活的影响。方法以胶原酶原位灌注消化加密度梯度离心法分离大鼠原代HSC,将siRNA以脂质体包裹,转染培养72h的原代HSC,设空白对照,收集孵育96h细胞,以Westernblot和免疫细胞化学染色法检测原代HSCCTGF及α-平滑肌肌动蛋白(α-SMA)基因表达。结果转染siRNA的原代HSCCTGF及α-SMA基因表达显著下调,与对照相比,CTGF蛋白下调(92±5)%(P<0.01);α-SMA蛋白表达分别下调(62±9)%(P<0.01,Westemblot方法)和(58±6)%(P<0.01,免疫细胞化学染色法)。结论siRNA能显著下调大鼠原代HSCCTGF基因表达,明显阻抑原代HSC培养激活,提示针对CTGF基因化学合成的siRNA具有防治肝纤维化的潜力。     

维生素E对大鼠肝星状细胞增殖及细胞外基质的影响

目的 观察维生素E（VE）对大鼠肝星状细胞（HSC）过氧化脂质和细胞外基质（ECM）产生的影响。方法 采用原位灌注方法分离大鼠HSC。结果 新分离的大鼠HC培养1w时具有增殖和ECM分泌的活性。VE可有效抑制大鼠HSC^3H-胸腺嗜啶（^3H-TdR）、^3H-脯氨酸（^3H-Pro）的掺入和Ⅲ型前胶原（PCⅢ）及透明质酸（HA）的分泌，VE作用后HSC产生丙二醛（MDA）下降，与分泌PCⅢ的抑制呈正相关。结论 VE可抑制大鼠HSC增殖和减少ECM的分泌。     

纤溶酶原激活物抑制剂-1 小干扰RNA对肝星状细胞生物学特性的影响

目的研究化学合成纤溶酶原激活物抑制剂-1（PAI-1）小干扰RNA（siRNA）对肝星状细胞（HSC）PAI-1基因表达及生物学特性的影响。方法将化学合成抗PAI-1 siRNA以Lipofectamine包裹，转染HSC—T6细胞，设阴性对照和空白对照，抽提细胞总RNA及蛋白质，收集培养上清液，应用逆转录-聚合酶链反应和免疫细胞荧光法检测细胞PAI—1表达。应用MTT法和流式细胞仪检测细胞增殖和细胞周期变化，应用酶联免疫吸附法检测培养上清液中Ⅰ、Ⅲ型胶原含量。结果转染siRNA的HSC—T6细胞PAI-1基因表达水平明显下调，HSC—T6细胞增殖抑制明显，48h和72h时培养上清液中Ⅰ型胶原表达水平显著减少，分别比阴性对照下降（20．71±8．40）％和（37．97±6．40）％（F=42．69。P=0．0001），Ⅲ型胶原含量于72h时降低（35．98±4．60）％（F=105．52，P=0．0001）。结论化学合成抗PAI-1 siRNA能高效抑制HSC—T6PAI-1表达，抑制HSC—T6细胞增殖，显著减少Ⅰ、Ⅲ型胶原的合成和分泌，具有预防及治疗肝纤维化的潜力。     

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.     

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...     

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 from patients with cirrhosis increases tissue plasminogen activator release from vascular endothelial cells in vitro

ABSTRACT— Aims/Background: In patients with severe liver disease, blood levels of many coagulation and fibrinolytic factors are lowered due to a diminished synthetic capability in the liver. Tissue plasminogen activator (t-PA) is synthesized by the vascular endothelial cells, however, and is increased in such patients. Methods: Amounts of t-PA secreted were determined by immunosorbent assay after plasma from patients with cirrhosis was added to cultured human umbilical vein endothelial cells to determine whether a plasma factor directly enhanced t-PA secretion from vascular endothelial cells. Results: Release of t-PA was significantly higher with exposure to plasma from patients with decompensated cirrhosis than when plasma from patients with compensated cirrhosis or normal subjects was used (p<0.01 and p<0.05, respectively). Plasminogen activator inhibitor 1(PAI-1) concentrations were measured similarly but did not differ among the three groups. Conclusions: Our results indicate that factors in plasma from patients with decompensated cirrhosis directly stimulate t-PA release from the vascular endothelial cells, while any increased PAI-1 release observed in comparable in vivo situations is probably an indirect response to an increase of t-PA or a result of impaired hepatic clearance.     

t-PA、PAI活性测定在早期糖尿病肾病临床中的意义

目的 探讨2型糖尿病（T2DM）患者及糖尿病肾病（DN）患者中尿白蛋白排泄率（UAER）与血浆组织型纤溶酶原激活物（t-PA）、纤溶酶原激活物抑制物（PAI）活性的相关性。方法 选择60例T2DM病人，根据UAER分为单纯糖尿病（DMa）组、微量白蛋白尿组（DMb）和临床蛋白尿组（DMc）。此外，还选择了30例健康人作为对照组。采用发色底物显色法测定血浆t-PA和PAI的活性，并对其相关性进行统计分析。结果 （1）对照组、DMa组、DMb组和DMc组血浆卜PA活性递减，PAI的活性递增，各组比较有显著性差异（P〈0．01）。（2）t-PA与UAER呈负相关（r=0．615，P=0．000），PAI和UAER呈正相关（r=0．721，P=0．000）。结论 DN早期即有纤溶活性低下；t-PA和PAI可能作为DN肾脏损害程度的佐证，对指导临床用药以缓解或延迟DN的发生具有重要意义。     

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。     

纤溶酶原激活物抑制物-1基因4G/5G基因型分布频率与心肌梗死和脑梗死相关性初步分析

目的研究我国汉族人纤溶酶原激活物抑制物-1基因启动子区-675位4G/5G(单鸟嘌呤核苷酸插入/缺失)基因多态性与心肌梗死和脑梗死的等位基因特异性的相关性.方法以等位基因特异性聚合酶链反应(AS-PCR)扩增56例心肌梗死患者,54例脑梗死患者,83例无关健康对照个体的基因组DNA,鉴定PAl-1 4G/5G基因型及分布频率,常规方法检验研究个体的主要临床和生化指标.结果PAI-1基因启动子区4G/5G基因多态性在心肌梗死,脑梗死患者组中的分布频率与对照组明显不同.在心肌梗死组中,4G/4G基因型分布频率(71.40%)比对照组(30.12%)显著增加(P＜0.001),杂合型4G/5G基因型分布频率(25.00%)比对照组(62.65%)明显降低;而在脑梗死组中,4G/4G,4G/5G基因型分布频率(分别为20.37%,55.56%)均比对照组(分别为30.12%,62.65%)低,5G/5G基因型明显增加(24.07%vs7.32%,P＜0.001).而且心梗组中血浆PAI-1活性水平随着4G等位基因的减少而降低;脑梗死组中,血浆PAI-1活性水平随着5G等位基因的升高而增加.心肌梗死、脑梗死患者组中的血浆PAI-1活性水平,甘油三酯水平和血糖水平都比对照组明显升高(分别为P＜0.001,P＜0.05,P＜0.001).结论本研究表明,中国汉族人PAI-1基因启动子区4G/5G基因多态性可能和心肌梗死、脑梗死发生的危险性相关,4G/5G基因多态性可能是一种重要的遗传性血栓性疾病的危险因子.     

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.     

Protection by α2-macroglobulin of tissue plasminogen activator against inhibition by plasminogen activator inhibitor-1

Tissue plasminogen activator (tPA) is widely used in the treatment of acute myocardial infarction (MI). However, its thrombolytic efficacy does not correlate with the dose administered. The interactions between tPA, α2-macroglobulin (α2-M), and plasminogen activator inhibitor-1 (PAI-1) were investigated both in vitro and in patients undergoing tPA therapy for MI in an attempt to identify variables that might affect the clinical efficacy of tPA.
Purified α2-M (5.4 mg/ml) protected 16.0% or 22.4% of tPA (12.5 IU/ml) activity from inhibition by PAI-1 at 4 or 8 IU/ml in vitro . Of nine patients treated with 5–20 mega IU of tPA for MI, the plasma activity of tPA remained increased for 15–30 min after the cessation of infusion in eight; the patient who failed to exhibit a persistent increase in tPA activity had a low plasma concentration of α2-M. Total tPA activity, derived from the area under the activity-versus-time curve (AUC), showed a significant inverse correlation with the ratio of the plasma PAI-1 activity to the plasma α2-M concentration. Total tPA activity did not correlate with plasma PAI-1 activity or plasma α2-M concentration alone. Results suggest that α2-M, by binding to tPA, protects the latter against inhibition by PAI-1.     

纤溶酶原激活剂抑制物的基因多态性与狼疮性肾炎肾小球的微血栓

目的：探讨纤溶酶原激活剂抑制物－1（PAI－1）基因启动子区－675bp4G/5G多态性与狼疮性肾炎（LN）肾小球微血栓形成的关系。方法：选取101例LN患者,所有患者均行经皮肾活检。其中46例患者伴有肾小球毛细血管袢内微血栓（T组）,55例患者不伴血栓（non-T组）。应用PCR－SLP法分析基因型。同时收集肾活检时各患者的临床资料。正常对照组包括128名健康成人。结果：①T组血清肌酐（SCr)水平显著高于non-T组,蛋白尿和血尿也较non-T组严重;但两组间血清抗心磷脂抗体阳性率无差异;②PAI－I基因4G／4G基因型及4G型等位基因与T组显著相关;LN中4G／4G型患者形成袢内血栓的相对风险率为OR＝2．96,95％CI：1．26－6．92。结论：LN肾小球微血栓的形成与PAI－I基因4G／5G多态性密切相关。     

Differential expression genes analyzed by cDNA array in the regulation of rat hepatic fibrogenesis.

PURPOSE: To analyze the gene expression pattern in rat hepatic fibrogenesis and further assess the role of some key genes during the pathological process. METHODS: Hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine or carbon tetrachloride (CCl(4)) injection subcutaneously in rats, and identification of the hepatic fibrosis related genes with cDNA microarray was performed. After some key genes up-regulated during the development of hepatic fibrosis were screened and confirmed, their effects on the function of the activated rat hepatic stellate cells (HSC) were assessed using the small interfering RNA (siRNA) technique. RESULTS: Using an Atlas rat cDNA array, a number of differentially expressed genes in fibrotic liver tissues were identified compared with non-diseased control. A total of 15 genes predominantly associated with the mitogen-activated protein kinase (MAPK) signal transduction pathway were upregulated in the fibrotic liver. Immunohistochemical study revealed that the expressions of both extracellular signal-regulated kinases (ERK) and ribosomal protein S6 kinase (RSK), two of the key genes in the MAPK pathway, were remarkably induced, which was closely correlated to that of collagen types I and III during the development of hepatic fibrosis. Transfection of siRNA targeting ERK1 mRNA (siERK1) into HSC led to a 66% and 72% reduction of ERK1 mRNA and protein expression, respectively. Furthermore, siERK1 exerted the inhibition of the proliferation of HSC, accompanied by the induction of HSC apoptosis and reduction of collagen types I and III. In addition, siERK1 abolished the effect of platelet-derived growth factor-BB on the proliferation of HSC. CONCLUSIONS: The present study provided strong evidence for the participation of the MAPK pathway in the pathogenesis of hepatic fibrosis. Selective targeting of ERK1 inhibitors to HSC might present as a novel strategy for the treatment of hepatic fibrosis.