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。     

Biolistic nuclear transformation of Saccharomyces cerevisiae and other fungi

Summary The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.     

Hirudin and Hirulog: Advances in antithrombotic therapy

Summary Over the past two decades, advances in the prevention and treatment of coronary artery disease, e.g., coronary angioplasty and coronary thrombolysis, have been coupled with the need for improved antithrombotic therapy to prevent acute, life-threatening thrombotic complications. Concurrently, studies on the pathophysiologic mechanisms for the arterial thrombotic process have demonstrated a central, mediating role for thrombin. Brought forward from its identification in the late 19th century, the leech protein hirudin emerged as a model for recombinant DNA engineering and protein-mimetic drug design. Recombinant hirudin (r-hirudin) and Hirulog are the resulting drug candidates which, as direct thrombin inhibitors, offer several potential advances in the management of acute thrombotic disorders. Clinical trials of these drug candidates have already provided evidence for clinical activity and tolerability in thromboembolic disease. Definitive evidence for efficacy is currently sought in large, controlled studies in the settings of coronary angioplasty, coronary thrombolysis and unstable angina.Hirulog antithrombin is a product of Biogen, Inc., Cambridge, MA, U.S.A.     

聚乙二醇修饰水蛭素的分离纯化与活性分析

用活化的聚乙二醇(PEG)对水蛭素进行化学修饰，质谱分析表明，修饰产物是修饰度不同的分子质量相差5000u的水蛭素的混合物。用离子交换层析和凝胶过滤层析对反应产物进行分离纯化，离子交换层析难以达到完全分离；而采用凝胶过滤层析方法可较好地分离修饰度不同的各组份。生物学活性分析表明，不同的PEG修饰度对水蛭素的活性具有显著影响，连接1～2个PEG的水蛭素保持了原有活性，而连接3个PEG的水蛭素活性显著下降。     

地高辛标记探针检测重组双功能水蛭素中的DNA残留量

为检测注射用重组双功能水蛭素半成品中的DNA残留量，应用地高辛标记重组双功能水蛭素工程菌DNA，并以此为DNA探针进行点杂交，同时做特异性及灵敏度实验，结果表明，该方法检测最低限值可达1pg,且重复性好，特异性强，操作较简便，可用于双功能水蛭素制备工艺的质控及半成品的检定。     

疏血通对髋部骨折患者凝血-纤溶功能影响的临床研究

目的:评估疏血通对髋关节手术患者术后凝血-纤溶系统的影响.方法:选择2002年7月～2003年4月髋部手术患者共57例,根据入院时间(单双日)分为治疗组(34例)和对照组(23例),手术前3d开始分别采用疏血通和低分子肝素治疗,共10d,于用药前、术前、术后第1天、术后第3天、术后第7天,测定白陶土部分凝血活酶时间(KPTT)、凝血酶原时间(PT)、组织型纤溶酶原激活剂(t-PA)、组织型纤溶酶原激活剂抑制剂(PAI)、D-dimer、血小板聚集率,怀疑有深静脉血栓(DVT)者行静脉造影检查.结果:两组治疗前后KPTT,PT明显延长;治疗组t-PA活性明显增强(P＜0.05),D-dimer升高,PAI无明显变化,并抑制血小板聚集.结论:疏血通在预防和治疗髋关节术后静脉血栓形成中,不仅有抗凝作用,而且能激活纤溶系统,抑制血小板聚集,是一种安全有效的药物.     

Efficacy of hirudin in reducing cardiovascular events in patients with acute coronary syndrome undergoing early percutaneous coronary intervention.

AIMS: Although hirudin is superior to unfractionated heparin for prevention of death, myocardial infarction, or refractory ischaemia in patients with non-ST-elevation acute coronary syndrome, it is not clear whether hirudin is also of benefit in acute coronary syndrome patients undergoing early percutaneous coronary intervention. METHODS AND RESULTS: In the OASIS 2 trial, 10 141 patients with non-ST-elevation acute coronary syndrome were randomized to 72 h of intravenous hirudin or unfractionated heparin. Percutaneous coronary intervention was performed at the discretion of the investigator. One hundred and seventeen patients underwent percutaneous coronary intervention within the first 72 h ("early percutaneous coronary intervention"). In patients undergoing early percutaneous coronary intervention, hirudin compared with unfractionated heparin was associated with a significantly lower incidence of death or myocardial infarction at 96 h (6.4% vs 21.4%, OR 0.30; 95% CI: 0.10-0.88) and 35 days (6.4% vs 22.9%, OR 0.25; 95% CI: 0.07-0.86). In the unfractionated heparin group, death or myocardial infarction was significantly higher at 35 days in patients undergoing early percutaneous coronary intervention compared with those managed conservatively (22.9% vs 7.3%, OR 3.14, P<0.001) but this early percutaneous coronary intervention-related hazard was not observed in hirudin-treated patients (6.4% vs 6.8%, OR 0.94 P=1.0). A time-dependent covariate for percutaneous coronary intervention was not significant in a Cox regression model, suggesting a similar treatment benefit with hirudin before and after percutaneous coronary intervention. After adjustment for percutaneous coronary intervention propensity, the benefits of hirudin remained significant. There were three major bleeds in patients undergoing early percutaneous coronary intervention, all in patients randomized to hirudin. CONCLUSION: In patients with non-ST-elevation acute coronary syndrome undergoing early percutaneous coronary intervention, a direct thrombin inhibitor such as hirudin may be more effective than heparin in reducing the incidence of ischaemic complications.     

Sustained bleeding from bites of hatchling leeches

Prolongation of haemostatic parameters in the host achieved by hatchlingHirudo medicinalis differs quantitatively but not qualitatively from those achieved by adult leeches. The duration and the rate of blood loss from the bite wounds, the gain in body weight of leeches and the duration of feeding each increase with successive feeding episodes. The mean duration of bleeding from bite wounds of hatchling leeches feeding for the first time on human volunteers was 43 min compared to 6 min from control incisions and 600 min in adult leeches. The mean ‘whole body’ concentration of hirudin in 2-week-old hatchlings was 10 antithrombin units (AT-U) compared to 285 AT-U in the head of each adult leech.     

水蛭素活性因子对S_（180）肿瘤及血管新生的影响

目的探讨水蛭素活性因子对S180肿瘤及血管新生的影响,为临床用药提供理论依据。方法 （1）S180肿瘤实验：取小鼠44只,随机分组,接种S180瘤细胞。模型对照组给予生理盐水,阳性对照组给予5-FU,实验用药组分别给予不同剂量的水蛭素活性因子,连续用药14天,第15天剥离肿块,比较各组肿瘤生长情况,并计算抑瘤率。（2）血管新生实验：取纯种鸡胚蛋,随机分组,恒温箱中孵育。分别吸取生理盐水、水蛭素活性因子不同浓度的药液,滴于滤膜载体上,将含药载体种植于鸡胚绒毛尿囊（CAM）中,密封后放入恒温箱内孵育96h,滴加甲醛,20m in后立体显微镜下观察药物抑制新生血管的生长情况,并用计算机分析系统对CAM进行扫描,记数血管VN、血管VD、血管VA的多少和大小。结果水蛭素活性因子大小剂量用药组对S180肿瘤具有明显的抑制作用,其抑瘤率分别为43.33%和40.67%。CAM观察发现,水蛭素活性因子大小剂量用药组血管分布稀疏、间距增大,含药载体周围血管减少。而模型对照组则血管分布密集、间距缩小、呈放射状排列。结论实验表明,水蛭素活性因子对S180肿瘤和CAM新生血管具有较强的抑制作用。     

BAEE法测定重组水蛭素的生物活性

目的建立重组水蛭素的生物活性测定方法。方法用BAEE(N-苯甲酰-L-精氨酸乙酯)作底物,通过测定反应体系中未被水蛭素抑制的凝血酶的量,来间接测定水蛭素抗凝血酶的活力。结果重组水蛭素的生物活性测定曲线在3.2~12.8 ATU之间线性关系良好,回归方程为Y=-0.034 8X+0.7,r=0.999 3。结论BAEE法经济、便捷、准确可靠、重现性好,适宜作为该品种的生物活性测定方法。