冠心病患者内皮祖细胞tPA和PAI表达的变化

目的探讨冠心病患者外周血中内皮祖细胞(EPC)的变化及其组织型纤溶酶原激活物(tPA)和纤溶酶原激活剂抑制剂(PAI)的表达。方法选择冠心病患者57例和对照组30例,提取内皮祖细胞进行数量和细胞集落的比较。利用ELISA法和底物发光法检测EPC分泌tPA和PAI的浓度和活性;用RT-PCR法检测EPC的tPA和PAI mRNA表达。结果冠心病患者EPC数量较对照组明显减少(23.1±1.8比56.7±2.4,P<0.05),形成细胞集落数(14.7±2.5比24.2±1.7,P<0.05)、细胞增殖能力也明显降低,冠心病患者EPC的tPA表达较对照组下降,PAI表达增强。结论冠心病患者外周血EPC数量减少和功能障碍可能在疾病的发生发展中起作用。     

Decreased fibrinolytic capacity and increased von Willebrand factor levels as indicators of endothelial cell dysfunction in patients with lupus anticoagulant

Summary Over a 6-year period, 10 patients with lupus anticoagulant activity were seen. A history of thrombotic disease was found in 6 patients, but only 3 had systemic autoimmune disease. Reduced fibrinolytic activity after venous occlusion was found in 9 subjects, but only 4 had high von Willebrand factor levels. These changes were unrelated to inflammatory activity, which was ruled out by normal serum protein electrophoresis in all but one case. Human brain thromboplastin dilution test was pathological in all subjects with depressed fibrinolytic activity. These two tests may prove to be of value to single out those LA patients with highest risk for development of thromboembolic disease.     

纤溶酶原激活物抑制剂-1反义RNA对主动脉内皮细胞部分功能的影响

目的 探讨纤溶酶原激活物抑制剂－1（plasminogen activator inhibior-1,PAI-1）反义RNA对离体培养的主动脉内皮细胞（endothelial cell,EC）PAI－1表达的作用及对血管内皮生长因子（vascular endothelial growth VEGF）的影响。方法 聚合酶链反应（PCR）手增PAI－1第2外显子,将PCR产物纯化克隆后连入真核细胞表达载体pcDNA3．1（－）,构建PAI－1反义RNA重组质粒。将pcDNA3．1－反义PAI－1重组质粒转染EC中。通过免疫组化、免疫印迹法（Westernblot）、双抗体夹心法（ELISA）检测细胞中PAI－1表达的改变,通过免疫荧光技术观察细胞中PAI－1表达量的变化对VEGF的影响。结果 转染后第3天,细胞中PAI－1含量为0．017ng/ml,细胞浆内代表VEGF的绿色荧光最弱,表明VEGF的表达也减少;第5天,PAI－1含量为0．093ng/ml,细胞浆内绿色荧光物质略增加。第7天,PAI－1含量为0．143ng/ml,接近正常值,VEGF的荧光染色接近于正常水平。结论 反义PAI－1RNA能有效阻断EC中PAI－1的蛋白合成,同时抑制剂细胞中VEGF的表达。     

Ethanol-induced apoptosis in polarized hepatic cells possibly through regulation of the Fas pathway

BACKGROUND: It has been noted that alcohol-related liver diseases can be associated with an increase in apoptotic hepatocellular death. Moreover, the promotion of hepatocyte apoptosis may be linked to signals emanating from death receptors, particularly Fas [CD95/apoptosis-inducing protein 1 (APO-1)]. In the present study, we utilized an in vitro hepatic culture model [hybrid of human fibroblast (WI 38) and rat hepatoma (Fao) cells, WIF-B cells] to study potential contributing mechanisms involved in hepatocellular apoptosis following ethanol administration. METHODS: WIF-B cultures (differentiated hepatic cells that efficiently metabolize alcohol) were treated with or without ethanol and specific inhibitors of alcohol metabolism and cysteine protease activity, followed by morphological and biochemical examination of proapoptotic parameters. RESULTS: The results of this work demonstrated that ethanol administration leads to an increase (45%-60%) in caspase-3 activity and that the induction of apoptosis was found to be linked to the metabolism of alcohol. Additionally, increases were observed in the activity of upstream initiator caspases (caspase-2 and caspase-8) that are directly related to membrane signaling events of death receptors such as Fas. Moreover, it was determined that the activation of caspase-3 could be blocked by the presence of a specific caspase-8 inhibitor, again linking death receptor-associated proteases to downstream effector caspase activity in alcohol-related death. Finally, ethanol administration was found to result in an increase in the amount of Fas protein present in the membrane fraction of the cell. The increase in membrane Fas protein indicates ligand-independent membrane targeting of Fas in the alcohol-treated cells that could potentially be a key signaling event in the induction of the proapoptotic caspase cascade. CONCLUSIONS: The data presented here indicate that alcohol metabolism induces apoptosis in WIF-B cells that occurs, in part, by mechanisms involving signals emanating from death receptors.     

人尿激酶原cDNA在牛内皮细胞中的表达

本实验将人的尿激酶原（Ｐｒｏ－ＵＫ）ｃＤＮＡ导人体外培养的胎牛主动脉内皮细胞（ＥＣ），得到了表达该基因的转化细胞，以期覆盖血管内支架或小口径人工血管（＜４ｍｍ）等移植物达到防止血栓形成，提高移植物通畅率的目的。利用磷酸钙盐沉淀法将重组逆转录病毒载体ｐＮ２－ＣＭＶ·ＵＫ导人包装细胞ＰＡ３１７，获得了含有重组逆转录病毒（Ｐｒｏ－ＵＫＲＮＡ序列）的培养上滑。用机械刮取胎牛主动脉内腔面分离ＥＣ，进行常规培养。用重组逆转录病毒感染７代以内的牛ＥＣ，并经Ｇ４ｌ８筛选得到抗性细胞。Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明ＰｒｏＵＫｃＤＮＡ已整合进ＥＣ基因组。以鼠抗人的Ｐｒｏ－ＵＫ单克隆抗体为一抗，作细胞免疫组化分析（Ｌ５Ａ８法），抗性细胞胞浆中出现阳性棕色颗粒，对照细胞为阴性结果。溶圈实验测得尿激酶原分泌量约为２３Ｕ／１０６细胞／２４小时。上述结果证明人Ｐｒｏ－ＵＫｃＤＮＡ已整合入牛ＥＣ基因组，表达产物具有免疫活性和纤溶酶原激活作用。     

Thromboxane production by perturbed bovine aortic endothelial cells in culture

Summary Bovine aortic endothelial cells in culture were incubated with endotoxin. The amount of thromboxane A₂ synthesized was then determined by a specific radioimmunoassay for thromboxane B₂. After a lag of several hours the cells changed their shape and parallel to the change in cell shape release of thromboxane B₂ occurred. At 24h the amount of thromboxane B₂ generated in response to endotoxin was 200-fold above baseline. Thromboxane B₂ generation could be blocked by aspirin and the specific thromboxane synthetase inhibitor UK 37248. The endotoxin effect was dependent on protein and RNA synthesis as evidenced by the inhibitory action of cycloheximide (1.5M) and actinomycin D (2m).     

Impaired fibrinolysis in the hemolytic-uremic syndrome of childhood

Summary Thrombotic obstruction of glomerular capillaries causes acute renal failure in patients with hemolytic-uremic syndrome (HUS). Recanalization of occluded vessels normally occurs by activation of the endogenous fibrinolytic system, mediated by plasminogen activators, which are stored and synthesized in the endothelial cells. However, endothelial injury is considered the primary event in the pathogenesis of HUS, and this may result in impaired fibrinolysis. In five children with HUS we performed a prospective study of plasminogen activator activity and two plasminogen activator antigens: tissue-type plasminogen activator and urokinase-type plasminogen activator before and after intravenous desmopressin. Plasminogen activator inhibitor type-1 antigen was also studied. In the acute stage of HUS plasminogen activating activity was low, in spite of elevated levels of total plasminogen activator antigens. This decrease of plasminogen activating activity was due to high levels of the plasminogen activator inhibitor. Improvement of fibrinolysis paralleled recovery from HUS. We conclude that decreased fibrinolysis is an important pathophysiologic feature of HUS.This study was presented in part at the 33rd annual meeting of the American Society of Hematology (Denver, Colorado, December 6–10 1991)(Blood 1991, Suppl 1: 853), and at the 22nd annual meeting of the German Society of Pediatrie Nephrology (Munster, FRG, March 12–14, 1992)     

小剂量尿激酶对不稳定型心绞痛患者内皮素及纤溶酶原活性的影响

目的:评价小剂量尿激酶对不稳定型心绞痛患者内皮源性血管活性物质内皮素 1、组织型纤溶酶原活性的影响,以探讨小剂量尿激酶治疗心绞痛的机制。方法:选择80例心电图负荷试验阳性、动态心电图出现缺血性改变的不稳定型心绞痛患者,静脉应用小剂量尿激酶3 d,于治疗前后分别测定内皮素 1、组织型纤溶酶原激活物及其抑制剂的变化。结果:与治疗前相比较,组织型纤溶酶原激活物升高,内皮素 1 及组织型纤溶酶原激活物抑制剂下降,其变化均具有显著性。结论:小剂量尿激酶可明显调节不稳定型心绞痛患者的内皮源性血管活性物质如内皮素 1、组织型纤溶酶原激活物及其抑制剂的活性。     

丁酸对人结肠癌细胞尿激酶型纤溶酶原激活剂及受体表达的影响

目的探讨丁酸对人结肠癌细胞尿激酶型纤溶酶原激活剂(u-PA)及其受体(u-PAR)表达的影响。方法将人传代结肠癌细胞株SW1116接种于不含或含不同浓度丁酸(2、3、4、7、10mmol／L)的培养基中，培养6、24、48、72h后，收集细胞用ELISA法测定胞质中u-PA和u-PAR的表达情况。结果随着培养基中丁酸浓度的增加和培养时间的延长，经丁酸处理的SW1116细胞表达的u-PA和u-PAR与对照组相比显著减少，当丁酸浓度≥4mmol／L和培养时问≥48h时，两者表达不再增加，反而呈逐步下降趋势。结论丁酸可抑制SW1116细胞u-PA和u-PAR表达。     

烟曲霉刺激后内皮细胞膜受体C型植物血凝素-1及Toll样受体的表达

目的 研究内皮细胞接触烟曲霉后表面受体树突状细胞相关性C型植物血凝素-1(Dectin-1)及Toll样受体2(TLR2)的表达变化,探讨其在免疫过程中的作用.方法 烟曲霉与人脐静脉内皮细胞共孵育,分不同时间(1、2、4及6 h)点收集细胞,流式细胞仪检测Dectin-1受体及TLR2表达的变化;提取细胞总蛋白,应用Western blot法检测细胞受体蛋白表达水平的变化;应用间接免疫荧光染色法观察各受体在细胞内外的分布情况.结果 (1)静息状态下,内皮细胞膜表面可见Dectin-1及TLR2表达,其平均荧光强度值分别为45和13.经烟曲霉刺激后,TLR2表达逐渐下降至18,Dectin-1表达先升高后下降,接触2 h后平均荧光强度值升至35,6 h后又降至18;(2)Western blot 法检测结果显示,内皮细胞经烟曲霉刺激后2 h Dectin-1蛋白表达最明显,4 h次之,6 h最低;而TLR2在各时间点均无明显变化;(3)共聚焦显微镜下可见部分烟曲霉出芽孢子和菌丝进入内皮细胞,细胞膜、孢子和菌丝表面均可见 Dectin-1表达;TLR2仅在细胞表面表达.结论 烟曲霉感染时,内皮细胞可表达Dectin-1及TLR2,这对识别是否为烟曲霉感染有重要价值.

Abstract：

Objective To observe the changes of TLR-2, Dectin-1 expression on endothelial cells,and to explore their role in the immune response after contact with Aspergillus fumigatus. Methods Aspergillus fumigatus and human umbilical vein endothelial cells were co-incubated. Cells were collected respectively after incubation for 0 h, 1 h, 2 h, 4 h and 6 h. TLR2 and Dectin-1 receptor expressions were detected by flow cytometry, and their protein was measured by Western blot. The distribution of the receptors in the cells were observed by immunofluorescence. Result TLR2 and Dectin-1 were expressed on the endothelial cell surface in quiescent condition. The mean fluorescence intensity of TLR2 on endothelial cells decreased from 45 to 13 stimulation by Aspergillus fumigatus, but the mean fluorescence intensity of Dectin-1increased from 13 to 35 in the first 2 hours and then decreased. By Western blot, the electrophoresis strip of Dectin-1 was most bright in 2 hours after contact with the fungus, and then decreased 4 and 6 hours. TLR2did not change significantly. Dectin-1 with fluorescent labeling was seen in spores and hyphae as well as in the cell membrane under confocal microscope. TLR2 was detected only on cell surface. Conclusion TLR2and Dectin-1 were expressed by endothelial cells, and may be useful in the identification of Aspergillus fumigatus.     

血管紧张素II对培养的人脐静脉内皮细胞产生纤溶因子的影响

摘要：　目的 探讨血管紧张素II(angiotensin II，Ang II)对内皮细胞纤溶功能的影响及缬沙坦的干预作用。方法 用酶消化法收集并培养人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)，将10-6，10-7，10-8，10-9mol/L Ang II分别与HUVECs共同孵育12 h；10-7mol/L Ang II和HUVECs分别孵育0，2，6，12，24，48 h；10-7mol/L Ang II和10-5，10-6，10-7，10-8mol/L缬沙坦(与HUVECs孵育12 h；10-6mol/L缬沙坦与HUVECs孵育12 h。用酶联免疫吸附法测定上清液中组织型纤溶酶原激活物(tissue plasminogen activator，tPA)和1型纤溶酶原激活物抑制剂(plasminogen activator inhibitor-1，PAI-1)抗原浓度。结果 Ang II呈浓度依赖性升高HUVECs的PAI-1水平，最大效应浓度10-7mol/L；10-7mol/L AngII对HUVECs作用，孵育2 h PAI-1含量即升高，12 h达高峰 198μg/L；缬沙坦呈浓度依赖性降低Ang II促HUVECs分泌PAI-1，缬沙坦最大效应浓度10-6mol/L； AngII和缬沙坦对HUVECs分泌tPA没有明显影响。结论 AngII通过促进HUVECs分泌PAI-1而降低纤溶活性；缬沙坦通过抑制AngII的促PAI-1分泌作用而提高纤溶活性，提示有助于动脉粥样硬化血栓性疾病的防治。     

缺血性心脑血管疾病患者血浆纤溶酶原激活物及纤溶酶原激活物抑制剂1的测定及临床意义

目的研究缺血性心脑血管疾病患者血浆尿激酶型纤溶酶原激活物及其受体、组织型纤溶酶原激活物及其抑制剂1的水平及意义。方法应用酶联免疫吸附试验测定急性脑梗死、急性心肌梗死及不稳定型心绞痛患者血浆尿激酶型纤溶酶原激活物及其受体、组织型纤溶酶原激活物及其抑制剂1的水平。结果(1)脑梗死患者急性期血浆尿激酶型纤溶酶原激活物轻度升高(P>0.05),恢复期明显回落(P<0.05),尿激酶型纤溶酶原激活物受体水平在急性期明显升高(P<0.01),恢复期进一步升高;血浆中组织型纤溶酶原激活物含量在急性期明显低于对照组(P<0.01),而纤溶酶原激活物抑制剂1含量则明显高于对照组(P<0.01),恢复期纤溶酶原激活物抑制剂1水平趋于正常,而血浆中组织型纤溶酶原激活物水平与对照组比较仍存在一定差异(P<0.05)。(2)急性心肌梗塞患者血浆尿激酶型纤溶酶原激活物受体水平急性期明显升高(P<0.05),恢复期进一步升高(P<0.01),尿激酶型纤溶酶原激活物水平均大致正常;急性期血浆中血浆中组织型纤溶酶原激活物及纤溶酶原激活物抑制剂1含量均明显高于对照组(P<0.01),恢复期明显回落,纤溶酶原激活物抑制剂1趋于正常,血浆中组织型纤溶酶原激活物水平仍高于对照组(P<0.05)。(3)不稳定型心绞痛患者急性期(入院时)血浆尿激酶型纤溶酶原激活物受体水平明显升高(P<0.01),恢复期(入院后二周)回落,但仍明显高于对照组(P<0.05),尿激酶型纤溶酶原激活物水平与对照组比较均未见明显差异(P>0.05);急性期血浆中组织型纤溶酶原激活物含量明显低于正常组(P<0.01),而纤溶酶原激活物抑制剂1含量略高于对照组(P>0.05),恢复期两者含量均趋于正常(P>0.05)。结论缺血性心脑血管疾病患者存在不同程度的凝血纤溶系统失平衡,对疾病的发生发展起重要作用。     

不同剂量阿托伐他汀对内皮前体细胞的动员

目的:探讨不同剂量的阿托伐他汀在不同时相对内皮前体细胞(EPC)的动员作用。方法:将雄性新西兰兔随机分为4组:正常对照组、阿托伐他汀2.5mg、5mg、10mg组。用药后1、2、3、4周分别测量外周血EPC含量。用流式细胞仪计数EPCs,PE-CD34／FITC-CD133双阳性细胞为EPC；荧光显微镜鉴定FITC-UEA-1/Dil-acLDL双染色阳性细胞为EPC。用药第3周测血清一氧化氮(NO)、血脂。结果:用药后1、2、3、4周四组外周血EPCs的曲线:正常对照组为一低水平基线；阿托伐他汀5mg组、阿托伐他汀2.5mg组(按作用从大到小排列)为锯齿样曲线,第二周最低,第三周最高,第四周较第三周低；阿托伐他汀10mg组与正常组接近,仅第四周增高(P<0.05)。阿托伐他汀5mg组对EPCs有持续动员作用,于第三周时效果最佳,约为第一周的3倍,是正常组的近20倍；阿托伐他汀2.5mg组在第三、四周有作用。在第四周,三个用药组EPCs均较正常组高,且用药三组间无统计学差异。与正常组相比,阿托伐他汀5mg组血清NO增高,阿托伐他汀10mg组NO降低。各组血脂均在正常范围内。结论:不同剂量阿托伐他汀对EPCs均有动员作用,其效果为:5mg组>2.5mg组>10mg组。阿托伐他汀5mg组在第三周效果最佳。阿托伐他汀对EPCs的动员可能与NO有关。     

尿激酶型纤溶酶原激活物受体在人脑胶质瘤中的表达及临床意义

应用流式细胞仪定量检测正常脑组织 (3例 )和胶质瘤 (32例 )标本中尿激酶型纤溶酶原激活物受体(u- PAR)含量 ,并探讨 u- PAR在人脑胶质瘤中的表达及其与临床各生物学参数的关系。结果显示 :u- PAR在人脑胶质瘤中的表达与病理分级呈显著正相关 (r=0 .83) ,而与病人性别、年龄、肿瘤部位、瘤体大小等因素无关。提示 u- PAR可作为判断人脑胶质瘤恶性程度和浸润潜能的一项参考指标。     

Studies on oral contraceptive-induced changes in blood coagulation and fibrinolysis and the estrogen effect on endothelial cells

Summary Blood coagulation (fibrinogen, thrombinantithrombin III complexes, TAT, and prothrombin fragment F₁₊₂) and fibrinolytic parameters [fibrin split-product D-dimer, tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor-1 activity (PAI-1), and plasmin-antiplasmin-complexes (PAP)] were evaluated in 16 women on low estrogen (EE) oral contraceptive (OC) therapy. Blood samples were taken before and between days 18 and 22 of the first, third, and sixth treatment cycle. Fibrinogen levels were found significantly elevated during OC treatment compared with pretreatment values, while TAT and also F₁₊₂ levels remained unchanged. Treatment-induced activation of fibrinolysis was documented by elevated D-dimer [pretreatment (pt): 172 ng/ml (range: 65–640 ng/ml), cycle 6 (c.6): 351 ng/ml (range: 93–960 ng/ ml),p<0.05)] and PAP [(pt: 46.6 ng/ml (13–220 ng/ml), c.6: 66.4 ng/ml (21–200 ng/ml),p<0.05] plasma levels. Among the fibrinolytic components a decrease in PAI-1 [pt: 10.8 ng/ml (2–56 ng/ml), c.6: 5.3 ng/ml (2.2–14.4 ng/ml),p<0.05] and an increase in t-PA activity [pt: 0.23 U/ml (0.17–0.45 U/ml), c.6: 0.33 U/ml (0.2–0.9 U/ml),p<0.05] were detected. Experiments with cultured human endothelial cells (EC) showed that EE influenced neither EC hemostatic regulatory activities (tissue factor, thrombomodulin) nor the secretion of the fibrinolytic components t-PA and PAI-1.