天然、重组水蛭素对随意皮瓣淤血模型血管内皮细胞生长因子的影响

背景：随意皮瓣移植后易发生皮瓣静脉淤血,甚至坏死,临床与动物实验证明水蛭素能够提高淤血皮瓣的成活率。 目的：观察水蛭素对大鼠随意皮瓣淤血模型成活保护作用。 方法：Wistar大鼠30只背部设计随意皮瓣10 cm×3 cm制成淤血模型,随机分为天然水蛭素组、重组水蛭素组和对照组,分别于皮下注射5 U天然水蛭素、5 U重组水蛭素和生理盐水。术后测定血管内皮细胞生长因子含量及血管内皮细胞生长因子mRNA表达量,计算皮瓣成活率。 结果与结论：天然水蛭素组与重组水蛭素组皮瓣成活率比对照组高(P< 0.05)。天然水蛭素组与重组水蛭素组比对照组新生毛细血管血管内皮细胞生长因子表达多(P < 0.05),且天然水蛭素组最多。提示天然、重组水蛭素均能够促进随意皮瓣血管内皮细胞生长因子表达,有利于新生血管的生成,能够提高随意皮瓣的成活率,且天然水蛭素效果优于重组水蛭素。     

天然与重组水蛭素对大鼠随意皮瓣淤血模型超氧化物歧化酶、丙二醛、内皮素变化的影响

背景：一般认为天然水蛭素能够提高淤血皮瓣的成活率与其抗血栓、抗凝血酶有关,但其抗炎、抗氧化作用的具体机制尚不清晰。重组水蛭素效果与天然水蛭素的差异也未有深入研究。 目的：探索天然、重组水蛭素对大鼠随意皮瓣淤血模型存活保护机制。 方法：Wistar大鼠30只,背部设计随意皮瓣10 cm×3 cm制成淤血模型。皮瓣随机分为天然水蛭素组、重组水蛭素组和对照组,均于术后即刻、第3,5天注射在距皮瓣末端1.5 cm和3.0 cm处分别注射5 U天然水蛭素、5 U重组水蛭素和相同剂量生理盐水。观察术后第7天的皮瓣成活率、皮瓣组织细胞形态学改变以及超氧化物歧化酶、丙二醛、内皮素水平。 结果与结论：与对照组相比,天然、重组水蛭素组皮瓣成活率高,皮瓣超氧化物歧化酶水平明显增多,内皮素、丙二醛水平减少。且天然水蛭素效果优于重组水蛭素。说明水蛭素能够提高随意皮瓣的成活率、超氧化物歧化酶的含量;减低皮瓣的坏死率、丙二醛、内皮素的含量。     

低分子肝素和重组水蛭素对兔耳静脉淤血皮瓣成活的影响

背景：低分子肝素和水蛭素都有活血化淤作用。  目的：观察局部注射低分子肝素或重组水蛭素对静脉淤血皮瓣成活的影响。 方法：在24只兔左耳背制作3 cm×6 cm静脉淤血皮瓣模型,随机分组：对照组皮瓣下多点注射生理盐水;肝素组皮瓣下多点注射低分子肝素625 U/mL,水蛭素组皮瓣下多点注射重组水蛭素1 U/mL。 结果与结论：①大体观察：术后水蛭素组和肝素组较对照组淤血程度轻,血肿消散快。②皮瓣成活面积百分比：水蛭素组、肝素组明显高于对照组(P < 0.01),水蛭素组和肝素组比较差异无显著性意义(P > 0.05)。③苏木精-伊红染色结果：对照组术后同期微血管淤血程度较水蛭素组和肝素组严重,微血管计数明显少于水蛭素组和肝素组(P < 0.05,P < 0.01),水蛭素组微血管计数明显高于肝素组(P < 0.01)。说明局部应用低分子肝素或重组水蛭素可较快改善皮瓣的淤血,提高皮瓣成活,重组水蛭素效果优于低分子肝素。      

噬菌体改组库的构建及高活性水蛭素变异体的筛选

目的： 运用DNA家族改组方法提高水蛭素的抗凝血酶活性.方法： 将水蛭素HV1、 HV2、 HV3基因等量混合, 用DNase I消化, 回收50 bp左右的片段, 再经无引物和有引物PCR扩增获得与原基因大小相同的改组分子, 构建噬菌体展示的水蛭素改组文库, 经凝血酶亲和淘选, ELISA筛选高活性的水蛭素变异体.结果： 经过DNA改组和噬菌体亲和筛选, 成功获得抗凝血酶活性提高的水蛭素变异体HV2-N47K.结论： 用DNA家族改组及亲和淘选技术能够筛选出高活性的水蛭素变异体蛋白, 这为其他分子的改组提供了借鉴.     

天然水蛭素水凝胶可改善随意皮瓣的存活

背景：随意皮瓣移植后易发生瘀血,甚至缺血坏死,临床与动物实验已经证明水蛭素能够提高皮瓣的存活率。目的：探讨天然水蛭素凝胶对大鼠随意皮瓣存活的影响。方法：制备天然水蛭素凝胶,用改良的Franz扩散池进行经皮渗透实验,使用Markwardt法进行水蛭素活性的检测。选用Wistar大鼠,在其背部设计随意皮瓣,面积为9 cm×3 cm;皮瓣随机分3组,分别涂抹生理盐水、天然水蛭素凝胶、空白凝胶基质。术后进行大体、组织病理学形态观察,第7天计算皮瓣存活率。结果与结论：体外实验：Markwardt法检测到天然水蛭素凝胶组的活性成分能透过皮肤,而空白凝胶组未检测到任何活性成分。体内实验：术后7 d,天然水蛭素凝胶组皮瓣坏死长度明显长于生理盐水组、空白凝胶基质组,差异有显著性意义(P < 0.05)。术后 3 d,天然水蛭素凝胶组真皮内毛细血管及微静脉红细胞瘀滞现象较生理盐水组、空白凝胶基质组明显减轻;术后7 d,天然水蛭素凝胶组皮下胶原及肉芽组织增生明显优于生理盐水组、空白凝胶基质组;术后7 d,天然水蛭素凝胶组皮瓣存活率高于生理盐水组、空白凝胶基质组,差异有显著性意义(P < 0.05)。结果表明天然水蛭素可渗透进入完整皮肤组织,局部应用天然水蛭素水凝胶同样对皮瓣移植后瘀血有改善作用,有利于新生血管的生成。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

重组RGD肽水蛭素改善大鼠冠脉微循环及其机制的研究

目的观察双重组RGD肽水蛭素对大鼠冠脉微循环障碍的疗效 ,并探讨其作用机理。方法健康雄性SD大鼠共62只 ,随机分为正常对照组 (n=6)、安慰剂组 (n=18)以及RGD肽水蛭素治疗组(n=36) ,后者根据所用RGD肽水蛭素剂量进一步分为1mg/kg和5mg/kg体质量2个亚组 ,每组各18只。RGD肽水蛭素于建模当日采用腹腔注射的方法给药 ,以注射生理盐水做为安慰剂。分别采用病理切片、超声心动图和彩色微粒子测量技术 ,观察各组大鼠心肌内微血栓和微梗死形成 ,以及心功能和心肌局部血流量变化的情况 ;采用免疫组化和RT -PCR的方法观察心肌组织内IL -18蛋白及mRNA的表达。结果与安慰剂组相比 ,1mg/kg/d及5mg/kg/d的RGD肽水蛭素可显著减少大鼠心肌内微栓塞数量以及局部梗死面积 (P<0.01) ,同时大鼠心功能LVEF和心肌局部血流量也明显得到改善 ;RGD肽水蛭素也显著下调大鼠心肌组织内IL -18蛋白和mRNA的表达 (P<0.05)。结论RGD肽水蛭素能明显改善大鼠因微栓塞所致的冠脉微循环障碍 ,可作为治疗冠脉微循环障碍的重要药物。其中抑制IL -18的分泌表达 ,可能是RGD肽水蛭素抗炎改善冠脉微循环的重要机制之一。     

水蛭素缓释,控释给经系统的研究进展

水蛭素是新型高效抗凝抗血栓药物，正被开发成不同缓释、控释系统，用于人体血栓的预防和治疗。本文将对几种常见的控释系统进行概述，特别地水蛭素一生物可降解一的支架的特点，功效以及对冠脉病变部位揎位释放进介绍，这种控释系统对ＰＴＣＡ术后的再栓塞具有很好的疗效。     

重组葡激酶-水蛭素融合蛋白的构建表达

背景：重组融合蛋白是根据凝血酶识别顺序将葡激酶与水蛭素串联而成的蛋白,具有双重功能,分子质量为23 ku,可在工程菌高密度发酵状态下获得高效表达。 目的：通过对工程菌进行高密度发酵,构建重组葡激酶-水蛭素融合蛋白纯化工艺,并探讨其构建可行性及表达价值。 方法：对工程菌进行高密度培养,并诱导表达重组葡激酶-水蛭素融合蛋白,对菌体进行离心收取,多次冻融后通过超滤、两部阴离子交换层析方法收集上清液,对重组葡激酶-水蛭素融合蛋白进行分离纯化,观察其纤溶比活性及在菌体中的高效表达价值。 结果与结论：发酵培养工程菌,并在第17小时时予以诱导。结果发现,诱导0.5 h时,便能观察到重组葡激酶-水蛭素融合蛋白表达,且发酵时间越长重组葡激酶-水蛭素融合蛋白表达量相应越大;至发酵20 h时,表达达到顶峰。菌体干质量为32.20 g/L,目的蛋白表达量约为1.48 g/L。经过纯化处理后,重组葡激酶-水蛭素融合蛋白纯度高达98%,纤溶比活性约为2.6×10⁴ IU/mg,回收活性的概率为56%。实验通过构建重组葡激酶-水蛭素融合蛋白纯化工艺,并分析其表达价值,提示该纯化工艺较为便捷,耗时较短,且能重复使用,可用于大规模生产。     

水蛭素缓释、控释给药系统的研究进展

水蛭素是新型高效抗凝抗血栓药物，正被开发成不同缓释、控释系统，用于人体血栓的预防和治疗。本文将对几种常见的控释系统进行概述，特别对水蛭素一生物可降解聚合的支架（ｓｔｅｎｔ）的特点，功效以及对冠脉病变部位的定位释放进行介绍，这种控释系统对ＰＴＣＡ术后的再栓塞具有很好的疗效。     

应用凝血酶偶联磁微粒技术对水蛭类药材中抗凝血酶活性的检测

水蛭是传统中药,它的主要药效功能之一是具有抗凝血酶活性组分。而在中药材及其制剂中能否检测出具有抗凝血酶活性,除与药材制取方法和制剂制备的工艺有关外,还可能与所含有的伪水蛭素含量有关。本文报道应用凝血酶偶联带有氨基末端的磁性微粒,从两种水蛭干品浸出液中分离纯化水蛭素,通过对抗凝血酶活性的检测,以求取对药材质量的评价,本法工艺简单、快速,亦可用于批量生产的初步纯化,为进一步精制提供基础。     

凝血因子Ⅺ--抗血栓形成的新靶点

近年来血栓性疾病发病率上升，严重危害人类的健康。目前临床使用的抗血栓药物存在出血的毒副作用。大量研究表明，凝血因子Ⅺ(factor Ⅺ，FⅪ)基因剔除或缺陷无出血倾向，却可以抑制闭塞性血栓形成。近年来开展的以FⅪ为靶点的抗血栓研究已取得了一定进展。本文就FⅪ作为抗血栓药物新靶点的依据，FⅪ在血栓形成中的作用及其作为抗血栓药物靶点的研究进展做一综述，以期为研究新的抗血栓药物提供思路。     

Effects of antithrombotic agents evaluated in a nonhuman primate vascular shunt model.

The effects of aspirin, cyproheptadine, dextran, dipyridamole, and sulfinpyrazone on thrombus deposition were determined. These antithrombotic agents were evaluated in a nonhuman primate model for thrombus generation that employed test devices exposed to blood in an arteriovenous shunt. Thrombus deposition on test devices was quantitated gravimetrically. Of the antithrombotic agents tested, cyproheptadine was found to be the most effective, and aspirin, dextran, and dipyridamole were each somewhat less effective. Sulfinpyrazone had only a slight antithrombotic effect. Ultrastructual studies of thrombus deposited in test devices showed that the various antithrombotic agents tested did not prevent completely the formation of fibrin, aggregation of platelets, or adhesion and spreading of platelets and leukocytes. This model for thrombus generation is felt to be a more efficient means for evaluating antithrombotic agents than previously described nonhuman primate models.     

Dermatan sulfate and the prevention of experimental venous thrombosis

Although the specific anticoagulant activity of dermatan sulphate is seventy times less than that of standard heparin, its venous antithrombotic activity, tested on a great number of experimental models, appears at gravimetric doses which are only seven fold higher. This antithrombotic activity is not correlated with the factor Xa inhibition, but is associated with thrombin generation inhibition and potentiation of heparin cofactor II. Meanwhile, others factors, still non entirely identified, i.e. like the release of endogenous tissue plasminogen activators, must probably be involved in the antithrombotic activity of dermatan sulphate. In contrast to heparin, dermatan sulphate possesses hemorrhagic properties only at doses which are forty times higher than the antithrombotic dose. These hemorrhagic properties seem associated with an inhibition of collagen induced platelet aggregation. Finally, the pharmacokinetic profile of dermatan sulphate after intravenous injection in the rabbit, is different from that of standard heparin, and close to that of low molecular weight heparins.     

Dynamics of thrombus formation on an artificial surface in vivo. Effects of antithrombotic agents.

A nonhuman primate model for in vivo evaluation of antithrombotic agents is described. In this model, the formation of a thrombus on a segment of Silastic tubing placed in the vena cava of a rhesus monkey is utilized to evaluate the effectiveness of antithrombotic agents. Thrombus formation in this model was found to occur rapidly, but this initial deposit quickly was followed by a reduction in thrombus weight. Eventually, after 2 hours of implantation of the test device, thrombus weight again increased and reached an apparent plateau. Three different antithrombotic agents were evaluated with this model. Warfarin therapy was found to decrease the thrombus weight in approximate proportion to its effect on the prothrombin time. Aspirin and dextran each produced a decrease in thrombus weight in 2 of 3 animals tested. Individual differences in response to thrombotic agents are apparent, but despite this, the model appears to offer advantages for in vivo evaluation of antithrombotic agents.     

An experimentally antithrombotic strawberry variety is also effective in humans

Prevention of arterial thrombotic diseases is of high priority in developed countries. As inappropriate diet is regarded as an important risk factor of thrombotic events, daily intake of an antithrombotic diet may offer a convenient and effective way of prevention. Earlier we used animal models of thrombosis to find fruits and vegetables with potential antithrombotic activity. Among various strawberry varieties tested, a particular variety (KYSt-4, Nohime) showed a significant antithrombotic effect. The aim of the present investigation was to extend this study to humans, by testing the experimentally active KYSt-4 and inactive KYSt-10 variety for effectiveness in humans after oral intake. Filtrates of strawberries were prepared and administered orally. Thrombotic status was tested by a novel global test (Gorog Thrombosis Test). The strawberry variety (KYSt-4; Nohime) which earlier inhibited experimental thrombosis showed antithrombotic effects in humans, while the experimentally inactive variety (KYSt-10) as well as the relevant control (water) were ineffective.     

The impact of antithrombotic therapy on neurosurgical emergency referral load

The authors sought to determine the impact of antithrombotic therapy on emergency referrals at one neurosurgical centre. All emergency telephone referrals over a 90 day period were carefully documented with particular attention paid to current antithrombotic medications and their indication. Details regarding age, gender, diagnosis, radiological findings and treatment were also recorded. 713 emergency referrals were documented in the data collection period. 174 (24.4%) patients presented with intracranial or spinal haemorrhage and 75 (43.1%) of these were on antithrombotic therapy, ranging in age from 46-94 years (mean 71.1 years) with 29 (31.8%) on warfarin, 43 (47.2%) on aspirin and 15 (16.4%) on clopidogrel alone or in combination with another antithrombotic agent. 17 (22.6%) had no documented indication for antithrombotic therapy (all of these were on aspirin therapy) and 9 (31%) of those on warfarin had an INR in excess of 3.5 on presentation. Almost one quarter of those on antithrombotic therapy who presented with a haemorrhagic complication had no obvious indication for such therapy. One third of those on warfarin were over anticoagulated.     

NOACS治疗房颤合并急性冠脉综合征的研究

心房颤动合并急性冠脉综合征的发病率逐年增高,其治疗方案越来越受到人们的关注。由于冠脉血栓和房颤血栓的形成机制不同,因此抗栓治疗的难点在于如何平衡出血和血栓风险,以减少并发症的发生。近年来,新型口服抗凝药物的上市为抗栓治疗提供了新的思路,本文将对新型口服抗凝药物在房颤合并急性冠脉综合征患者抗栓治疗方面的相关研究进行综述。     

房颤伴ACS患者经皮冠状动脉介入术后抗栓研究

随着人口老龄化的加剧,心房颤动（房颤）患者越来越多,房颤伴有血栓风险,死亡率和致残率增高。部分房颤患者因合并急性冠脉综合征（ACS）需行经皮冠状动脉介入术（PCI）治疗,术后需针对房颤和ACS血栓形成的不同机制进行联合抗栓,但是如何平衡出血和血栓风险进而选择合适的策略仍是临床中一项极大的挑战。本文对近年来房颤伴ACS患者PCI术后抗栓治疗的有效性和安全性相关研究进行综述,以期指导临床个体化抗栓治疗方案的选择。     

Antithrombotic treatment of acute coronary syndrome

After we have become aware that arteriosclerosis provokes thrombotic and thromboembolic process, which is the most common cause of occlusion of coronary arteries, in the past 30 years significant improvements have been made in antithrombotic treatment of acute coronary syndrome. Antithrombotic therapy of acute coronary syndrome has been explored in few directions. These are thrombolytic multidrug therapy, anticoagulant therapy with nonfractional and low molecular heparin, therapy with glycoprotein IIb/IIIa receptor antagonists and antiaggregation therapy with acetylsalicylic acid and tienopyrid. Research results and clinical experience point to a conclusion that antithrombotic therapy is the cornerstone in the mariagement of acute coronary syndrome. In this paper, results of major studies and main antithrombotic treatment guidelines in acute coronary syndrome accepted by international professional associations are presented.     

Pharmacological effects of magnesium on arterial thrombosis--mechanisms of action?

At present no consensus exists on the role of magnesium in acute myocardial infarction and this is primarily due to conflicting results from recent clinical trials. The ISIS-4 trial clearly showed that magnesium infusion is without benefit when given after thrombolysis or many hours after symptom onset. In contrast, the LIMIT-2 study provided strong evidence that early magnesium administration, given before any thrombolytic therapy protects the myocardium and improves long-term survival. Debate on the interpretation of the trial results is still ongoing, and is particularly focused on the time-dependency of magnesium, which has emerged from recent experimental studies. Candidate mechanisms, by which magnesium might modify the outcome of acute myocardial infarction, includes antiarrhythmic properties, improved coronary perfusion and haemodynamics, protection of the ischaemic myocardium, and an antithrombotic effect. So far animal models have shown a time-dependent effect of magnesium when given for both myocardial protection in experimental ischaemia-reperfusion injury and for antithrombotic purposes. Additional clinical trials are warranted and these must be carefully designed and implemented in the light of the laboratory evidence now available. In the present review magnesium's antithrombotic properties is discussed with respect to its effect on platelets, coagulation, fibrinolysis, and endothelial mediators with vasodilating and antithrombotic qualities. Observations from studies in patients with preeclampsia, another clinical condition where platelet hyperreactivity is well-recognized and where magnesium therapy is well-established, are briefly discussed in the present paper.