内皮型一氧化氮合酶基因转染对移植人工血管内膜增生的作用*★

目的：平滑肌细胞增殖移行和血小板激活导致血栓形成是移植血管再狭窄的主要原因，一氧化氮可以抑制上述生物反应，但内皮型一氧化氮合酶基因转染是否抑制种植了平滑肌细胞的人工血管内膜增生还未得到证实。实验拟进一步观察内皮型一氧化氮合酶基因转染对种植平滑肌细胞的人工血管内膜增生的影响。 方法：实验于2006-04/2007-05在西安交通大学医学院中心实验室及分子生物学实验室完成。①实验材料：1月龄新西兰大白兔1只，用来获取平滑肌细胞。成年新西兰大白兔18只，随机数字表法分成3组，每组6只。正常细胞组移植未种植细胞的人工血管；LacZ转染组移植种植转染lacZ的平滑肌细胞的人工血管，内皮型一氧化氮合酶转染组移植种植内皮型一氧化氮合酶的平滑肌细胞的人工血管。②实验方法：构建含有报道基因lacZ和内皮型一氧化氮合酶基因的假型反转录病毒载体小鼠白血病病毒/疱疹性口炎病毒G糖蛋白，并转染平滑肌细胞。将转染了基因的细胞种植在人工血管上，并用血管旁路移植的方法植入兔腹主动脉。③实验评估：测定转染内皮型一氧化氮合酶基因及LacZ基因细胞培养上清中一氧化氮含量。血管植入30，100 d 后X-gal染色及苏木精-伊红染色观察人工血管上的平滑肌细胞，同时显微镜下测量每段血管内膜增生的厚度。 结果：纳入成年新西兰大白兔18只，均进入结果分析。①内皮型一氧化氮合酶转染组一氧化氮含量明显高于未转染的正常细胞组（P < 0.05)。平滑肌细胞转染lacZ基因后经X-gal染色，倒置显微镜下可见转染了基因的细胞被染成蓝色。②血管植入30 d，与正常细胞组比较，LacZ转染组和内皮型一氧化氮合酶转染组内膜厚度差异无显著性（P > 0.05）；100 d后，内皮型一氧化氮合酶转染组内膜厚度与正常细胞组无明显差异，与LacZ转染组相比较，差异显著（P < 0.05）。 结论：内皮型一氧化氮合酶基因转染抑制了种植平滑肌细胞的人工血管内膜增生。     

组织型纤溶酶原激活物及其抑制物与脑血管疾病

组织型纤溶酶原激活物 (ｔｉｓｓｕｅｐｌａｓ ｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ ,ｔ -ＰＡ)及其抑制物(ｔｉｓｓｕｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒｉｎｈｉｂｉｔｏｒ -1,ＰＡＩ - 1)是纤溶系统的二个重要组成部分。目前对二者的结构、特性以及与脑血管疾病的关系尚未完全阐明。缺血性脑血管病患者血浆中二者的活性、抗原含量测定甚至出现了矛盾的结果。1　ｔ -ＰＡ、ＰＡＩ- 1在纤溶系统中的作用　　纤溶酶原激活物 (ＰＡ)即组织型纤溶酶原激活物 (ｔ -ＰＡ)和尿激酶型纤溶酶原激活物 (ｕ -ＰＡ)以及它们的抑制物在纤维蛋白…     

上海地区汉族人群组织型纤溶酶原激活物基因多态性与脑梗死的关系

目的：探讨上海地区汉族人群组织型纤溶酶原激活物基因（TPA）多态性与脑梗死的关系。方法：采用PCR及基因测序技术检测157例急性脑梗死患者及169例对照组TPA-7351位点的多态性。应用非条件Logistic回归模型，调整混杂因素后，分析各基因型与急性脑梗死发生的关系。结果：在急性脑梗死组C／C基因型、C／T基因型和T/T基因型者分别为0．42、0．45和0．13。对照组依次分别为0．44、0．47和0．09。经调整混杂因素后，与C／C基因型相比，T/T基因型发生急性脑梗死的危险性显著升高（P〈0．05）；分层分析发现T/T基因型主要与腔隙性脑梗死的发病风险有关（P〈0．05）。结论：在上海地区汉族人群中，TPA-7351C／T基因多态性与腔隙性脑梗死发病有关。     

急性脑梗死患者尿激酶型纤溶酶原激活物及受体的变化

目的探讨急性脑梗死患者血浆尿激酶型纤溶酶原激活物(uPA)及受体(uPAR)的变化和临床意义。方法对66例急性脑梗死患者和30例健康者应用酶联免疫吸附双抗体夹心法(ELISA法)定量测定血浆uPA和uPAR的水平。结果与正常对照组相比,急性脑梗死组uPA含量(4756±632)ng/L,uPAR含量(3102±256)ng/L,明显高于正常对照组的uPA(1075±244)ng/L和uPAR(877±216)ng/L,差异有显著性(P<0.01)。随急性脑梗死患者病情严重程度的加重,血浆uPA和uPAR含量增高越明显。结论急性脑梗死患者血浆uPA和uPAR的水平明显升高,且与急性脑梗死患者的病情密切相关。     

水蛭提取液对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物和纤溶酶原激活剂抑制物1的影响

目的探讨水蛭提取液(HEL)对培养的大鼠脑皮质微血管内皮细胞分泌组织型纤溶酶原激活物(tPA)、纤溶酶原激活剂抑制物1(PAI-1)的影响。方法建立大鼠大脑皮质微血管内皮细胞培养实验模型。MTT法筛选HEL的有效浓度。检测培养上清液的tPA、PAI-1含量与活性变化,RT-PCR检测经HEL治疗组与生理盐水对照组处理后的微血管内皮细胞tPA与PAI-1的表达,免疫组化检测两组微血管内皮细胞tPA的表达。结果 HEL在一定浓度范围内(0.25～1mg/μl)可促进微血管内皮细胞的生长,有剂量依赖关系(P<0.05)。HEL治疗组较生理盐水对照组能促进培养的大鼠脑皮质微血管内皮细胞分泌tPA,同时提高其活性,促进tPA mRNA的表达及tPA免疫活性表达,且呈剂量依赖性表达增强(P<0.01)。结论 HEL在体外能激活内源性纤溶系统。     

组织型纤溶酶原激活物及其抑制物与缺血性脑血管病

组织型纤溶酶原激活物(tissue type plasminogen activator,tPA)及其抑制物(plasminogen activator inhibitor-1,PAI-1)是纤溶系统的两个重要组成部分.tPA可使纤溶酶原转化为纤溶酶,PAI-1能够调节tPA的活性,两者共同作用调节纤溶过程.重组组织型纤溶酶原激活物(r-tPA)目前在临床上已经得到较广泛的应用,其能够溶解血栓、明确减轻患者的神经功能缺损已得到充分的肯定.     

急性脑血栓形成患者脑脊液组织型纤溶酶原激活物及其抑制物含量测定的意义

目的 本文通过观察急性动脉硬化性脑血栓形成患者脑脊液组织型纤溶酶原激活物(t-PA)及其抑制物(PAI-1)的抗原含量,以探讨急性脑血栓形成患者的纤溶活性及其临床意义。方法 采用双抗体夹心固相酶联免疫吸附法(ELISA)检测31例患者脑脊液t-PA及PAI-1抗原含量,与20名对照组脑脊液进行比较。结果 急性脑血栓形成组脑脊液t-PA、PAI-1含量均显著高于对照组。结论 说明急性脑血栓形成患者纤溶活性明显下降,t-PS及PAI-1参与了脑血栓形成之病理过程;t-PA及PAI-1抗原含量是反映体内纤溶活性的2个重要指标。     

急性脑梗死超早期重组组织型纤溶酶原激活物溶栓治疗的临床研究

目的:探讨发病6h内急性脑梗死给予重组组织型纤溶酶原激活物(rt-PA)溶栓治疗的疗效及并发症,并分析预后相关因素。方法:共收集本院2001-2005年70例溶栓治疗的急性脑梗死病例,其中52例静脉溶栓,18例动脉溶栓,分析比较两组病例溶栓前后及3个月随访的ESS评分及Barthel指数结果;同时分析与预后相关的因素。结果:静脉和动脉溶栓组溶栓前及溶栓30min后ESS评分及Barthel指数迅速增加,溶栓前后分值有显著差异。1个月内颅内出血率为5.77%(静脉组)和16.67%(动脉组)。3个月时ESS评分及Barthel指数较溶栓后30min的评分有显著改善。结论:6h内动脉、静脉溶栓治疗均安全有效。     

静脉低剂量重组组织型纤溶酶原激活物治疗短暂性脑缺血发作的临床对照研究

目的观察短暂性脑缺血发作(TIA)患者采用静脉低剂量重组组织型纤溶酶原激活物(rt-PA)治疗后血浆t-PA、PAI-1在不同时间点的变化特点,评价其疗效及安全性。方法纳入试验的TIA住院患者42例,根据患者是否接受静脉低剂量rt-PA治疗分为rt-PA治疗组和阿司匹林对照组(每组21人),治疗组患者入院第1 d、2 d、3 d给予rt-PA 20 mg,静滴,1次/d,3 d后改为阿司匹林100 mg,口服,1次/d;对照组患者入院后即给予阿司匹林100 mg,口服,1次/d。两组患者均住院观察2周,有卒中危险因素者给予相应治疗。结果(1)治疗组与对照组患者血浆t-PA水平均高于正常,但治疗组患者的t-PA在第1 d和第3 d明显高于入院时水平和对照组,差别均有显著性意义(P＜0．05);治疗组与对照组血浆PAI-1水平间差别无显著性意义,但均高于正常水平。(2)治疗组患者第1 d TIA发作控制率为80．96%,对照组为66．67%。治疗组有效控制率为95．24%,对照组为90．48%。(3)治疗组未见主要不良事件发生。结论(1)静脉低剂量rt-PA治疗TIA可有效提高纤溶系统活性,抑制血栓形成;(2)静脉应用低剂量rt-PA是控制TIA急性发作的有效方法之一,且起效快、副作用小、安全性高。     

Regulation by nitric oxide of endotoxin-induced tissue factor and plasminogen activator inhibitor-1 in endothelial cells

The increase in nitric oxide (NO) production in lipopolysaccharide (LPS)-induced sepsis is thought to contribute to the development of shock. However, NO could also play an antithrombotic role. Little is known about the modulating effect of NO on the endothelial overexpression and production of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) occurring in endotoxemia. We analyzed the effect of N(G)-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases, and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, on the expression and synthesis of TF and PAI-1 by LPS-challenged human umbilical vein endothelial cells (HUVEC): L-NAME enhanced the increase in TF mRNA and antigen levels (P <0.05) observed in LPS-treated HUVEC; SNAP down-regulated the LPS-induced TF increment (p <0.05). However, no effects of NO on regulation of the LPS-dependent increase in PAI-1 could be seen. Thus, NO could play an antithrombotic role in sepsis by down-regulating the endothelial overexpression and production of TF.     

内皮型一氧化氮合酶基因转染对血管损伤后新生内膜增生的影响*

背景：一氧化氮能够抑制血管平滑肌细胞的迁移和增殖,而一氧化氮合酶是其合成的关键酶,有关一氧化氮合酶基因体内转染对平滑肌细胞及动脉粥样硬化血管损伤后内膜增生影响少有报道。 目的：观察内皮型一氧化氮合酶 (endothelial nitric oxide synthase,eNOS)基因体内局部转染对动脉粥样硬化大鼠血管损伤后新生内膜增生的抑制作用。 方法：建立动脉粥样硬化Wistar大鼠颈动脉球囊损伤模型,建模后随机分成空白对照组、AdCMV-lacz对照组和AdCMV-eNOS组,分别将PBS,AdCMV-lacz和AdCMV-eNOS体内转染至以上3组大鼠的损伤血管壁。转染2周后培养并鉴定损伤局部平滑肌细胞,并用RT-PCR法检测各组损伤及转染后血管平滑肌细胞eNOS mRNA的表达,同时观察转染后不同时期新生内膜增生的影响。 结果与结论：AdCMV-eNOS组的颈总动脉血管平滑肌细胞可表达eNOS mRNA。3组大鼠转染后1和3个月,AdCMV-eNOS组内膜/中膜面积比值低于空白对照组和AdCMV-lacz对照组(P < 0.01)。结果显示,eNOS基因体内转染损伤后血管可以抑制血管新生内膜增生,减少再狭窄发生率。     

Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells

The effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.     

Increased levels of circulating endothelial progenitor cells and circulating endothelial nitric oxide synthase in patients with gliomas

OBJECTIVE: Gliomas are among the highest vascularized tumors. We hypothesized that patients with gliomas have increased levels of circulating endothelial progenitor cells (EPCs) and circulating endothelial nitric oxide synthase (eNOS). METHODS: The fraction of EPCs was quantified by fluorescence-activated cell sorter analysis using anti-CD34, -CD133 and -KDR (kinase insert domain receptor) monoclonal antibodies in unselected peripheral blood samples of 32 patients with gliomas. Control groups included 47 patients with other central nervous system tumors or diseases, 10 patients with recent ischemic strokes, and 19 healthy blood donors. The circulating eNOS concentration of plasma was measured by a colorimetric assay in the same samples. In addition, CD34(+)CD105(+) KDR(+) and CD34(+)CD146(+)KDR(-) cell fractions were measured. RESULTS: The percentage of CD34(+)CD133(+)KDR(+) EPCs in the blood of glioma patients is significantly greater than that in the blood of patients with other central nervous system tumors or diseases (p = 0.003), stroke patients (p = 0.005), or healthy donors (p = 0.013). The plasma eNOS concentration is also significantly greater in glioma patients compared with each of the control groups (p < 0.001 for all groupwise comparisons). No significant differences in the levels of the EPCs or eNOS between any of the control groups were demonstrated. In the glioma patients, the level of eNOS correlated with the fraction of CD34(+)CD105(+)KDR(+) cells (r = 0.748; p = 0.008). INTERPRETATION: The data are suggestive of increased mobilization of EPCs contributing to neoplastic vasculogenesis in glioma. The increased levels of EPCs and eNOS in the peripheral blood of glioma patients trigger further investigations as to their value as independent parameters for use in clinical practice.     

Fluvastatin inhibits basal and stimulated plasminogen activator inhibitor 1, but induces tissue type plasminogen activator in cultured human endothelial cells

The effects of fluvastatin, a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitor, on the biosynthesis of tissue plasminogen activator (t-PA) and of its major physiological inhibitor (plasminogen activator inhibitor type 1, PAI-1) were investigated in cultured human umbilical vein endothelial cells (HUVEC). Fluvastatin (0.1 to 2.5 microM), concentration-dependently reduced the release of PAI-1 antigen by unstimulated HUVEC, subsequent to a reduction in PAI-1 steady-state mRNA levels and de novo protein synthesis. In contrast, it increased t-PA secretion. The drug also reduced PAI-1 antigen secreted in response to 10 microg/ml bacterial lipopolysaccharide (LPS), 100 U/ml tumour necrosis factor alpha (TNFalpha) or 0.1 microM phorbol myristate acetate (PMA). Mevalonate (100 microM), a precursor of isoprenoids, added to cells simultaneously with fluvastatin, suppressed the effect of the drug on PAI-1 both in unstimulated and stimulated cells as well as on t-PA antigen. Among intermediates of the isoprenoid pathway, all-transgeranylgeraniol (5 microM) but not farnesol (10 microM) prevented the effect of 2.5 microM fluvastatin on PAI-1 antigen, which suggests that the former intermediate of the isoprenoid synthesis is responsible for the observed effects.     

Agmatine inhibits matrix metalloproteinase-9 via endothelial nitric oxide synthase in cerebral endothelial cells

OBJECTIVES: Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane of brain vessels to promote cell death and tissue injury. We previously showed that agmatine has a neuroprotective effect on neurons against ischemic injury. In the present study, we investigated the effect of agmatine on the expression of MMPs and nitric oxide (NO) production in cerebral endothelial cells (CECs) after oxygen-glucose deprivation (OGD)-reperfusion injury and its potential association with endothelial nitric oxide synthase (eNOS). METHODS: Primary cultured endothelial cells from murine brain and bEnd.3 cells were subjected to OGD-reperfusion injury. Protein and mRNA levels of both MMP-2 and MMP-9 were determined by immunocytochemical analysis, Western blot and RT-PCR. Protein levels of eNOS were evaluated by Western blot in the CECs. The production of NO was measured using the Griess reagent. RESULTS: Agmatine attenuated the expression of MMP-2 and MMP-9 induced by ischemic injury at the protein and mRNA level, while agmatine increased the expression of eNOS directly. NO production was decreased in CECs after similar insult and was increased by agmatine treatment. In the presence of a nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), the expression levels of MMP-2 were decreased, but the expression of MMP-9 was not decreased by agmatine administration. However, NO production was suppressed by a non-specific NOS inhibitor in the agmatine treatment group. CONCLUSION: Our study supports that the down-regulation of MMP-9 by agmatine runs parallel to the up-regulation of eNOS and the maintenance of functional NO release.     

Specificity of the thrombin-induced release of tissue plasminogen activator from cultured human endothelial cells

The addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of alpha-thrombin, promoted tPA release but was less effective than alpha-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of gamma-thrombin 20 times greater than alpha-thrombin was required. The response to Factor Xa was similar to alpha-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active alpha-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.