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10cm×3cm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1.5cm和3.0cm处分别注射5U天然水蛭素、5U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。     

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10cm×3cm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5U天然水蛭素、5U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、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10cm×3cm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5U天然水蛭素、5U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。     

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3cm×6cm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625U/mL,水蛭素组皮瓣下多点注射重组水蛭素1U/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。     

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。     

中药水蛭素对荷瘤鼠超氧化物歧化酶及丙二醛水平的影响

目的:从抗氧化角度探讨中药水蛭素对荷瘤鼠血清超氧化物歧化酶(SOD)及丙二醛(MDA)水平的影响.方法:取小鼠44只,右腋下接种S-180瘤细胞,随机分为4组,模型对照组给予生理盐水,阳性对照组给予5-氟尿嘧啶,实验组分别给予不同剂量的中药水蛭素,连续用药14 d.于第15天取血,分离血清,检测SOD和MDA的含量.结果:中药水蛭素大小剂量组血清SOD含量明显升高,MDA的含量明显下降.结论:中药水蛭素能提高荷瘤鼠清除自由基的能力.     

灯盏花素注射液干预大鼠随意超长皮瓣成活

背景:灯盏花素能影响微血管舒张、减少缺血再灌注的损伤、降低血液黏滞性、对抗氧自由基损伤等,可能在皮瓣移植后治疗中起重要作用.目的:探讨灯盏花素对大鼠超长比例皮瓣成活的影响和可能的机制.方法:30只SD大鼠随机分为灯盏花素组、丹参组和对照组.在大鼠背部设计制备一蒂在尾端的8 cmx2 cm的超长全厚随意皮瓣,原位回植.灯盏花素组和丹参组在术后立即及之后每日分别腹腔内注射灯盏花素注射液4.5mL/(kg·d)及丹参注射液1.8 mL/(kg·d),对照组给予1 mL生理盐水.术后7 d,观察皮瓣存活情况,对皮瓣远、中及近段进行组织学观察;取大鼠皮瓣中轴距蒂部3 cm处组织,检测超氧化物歧化酶、丙二醛和一氧化氮水平.结果与结论:术后7 d,灯盏花素组和丹参组皮瓣存活率高于对照组(P＜0.05),其中灯盏花素组皮瓣存活率较高.灯盏花素组和丹参组近、中及远段组织水肿、坏死及炎症细胞浸润较对照组轻,其血管、成纤维细胞增生均明显优于对照组.灯盏花素组和丹参组超氧化物歧化酶及一氧化氮含量高于对照组(P＜0.05),其中灯盏花素组最高;而灯盏花素组和丹参组丙二醛含量低于对照组,灯盏花素组最低.说明腹腔注射灯盏花素注射液对大鼠随意超长皮瓣成活有明显促进作用,作用机制可能与扩张血管、改善循环、清除自由基及降低脂质过氧化反应有关,其作用优于丹参注射液.     

肾上腺髓质素真核表达载体对皮瓣缺血再灌注损伤的影响

背景:肾上腺髓质素可在心、脑、肾、肝等器官的缺血再灌注过程中起保护作用.目的:观察肾上腺髓质素真核表达载体对大鼠腹部皮瓣缺血再灌注损伤的影响及其作用机制.方法:构建大鼠肾上腺髓质素真核表达载体,将SD大鼠随机分假手术组、模型组、维拉帕米组、重组质粒组.重组质粒组腹部皮内注射肾上腺髓质素真核表达载体,其余各组注射等量生理盐水,各组注射4次后建立缺血再灌注模型,维拉帕米组于皮瓣内注射维拉帕米.结果与结论:与模型组相比,重组质粒组皮瓣组织中丙二醛、血管内皮素1的含量分别降低了38.50%(P < 0.01),54.73% (P < 0.01),超氧化物歧化酶的含量增加了50.67%(P < 0.05).皮瓣组织学检查结果显示重组质粒组皮瓣炎性细胞浸润与水肿程度也较模型组明显减轻.结果证实肾上腺髓质素真核表达载体对大鼠皮瓣缺血再灌注损伤具有保护作用,其机制与减轻脂质过氧化及减少炎症细胞浸润有关.     

大鼠背部超长原位回植皮瓣成活:人参皂甙Rb1影响的验证

背景:国内外将人参皂甙Rbl应用于心、脑、肺、肾及肝损害的保护与治疗的报道较多,但是将人参皂甙Rbl用于皮瓣的研究还未见报道.目的:观察腹腔注射人参皂甙Rbl对大鼠背部超长宽比例随意皮瓣成活的影响.方法:将健康成年SD大鼠以数字表法随机分为实验组及对照组.于大鼠背部设计制备蒂部在尾部的超长皮瓣80 mmx20 mm(长宽比4:1),原位回植.术中即刻、术后第1-5天腹腔注射2 mg/kg人参皂甙Rbl生理盐水溶液,对照组腹腔注射等量生理盐水.术后1 d取材测定皮瓣一氧化氮和丙二醛含量,术后10 d测定皮瓣存活率,取皮瓣近、中、远段行苏木精一伊红染色观察.结果与结论:术后1 d实验组皮瓣一氧化氮含量高于对照组(P<0.01),丙二醛含量低于对照组(P<0.01).术后10 d,实验组皮瓣成活率明显高于对照组(户<0.001 o组织学观察显示,实验组水肿及炎性细胞浸润较对照组明显减轻,纤维增生较对照组明显.结果提示罔手术期腹腔注射人参皂甙Rbl能明显改善大鼠皮瓣血供,提高皮瓣的成活面积和成活率,其作用机制可能与促进内皮细胞分泌释放NO、清除氧自由基、降低脂质过氧化产物、促进皮瓣血管新生有关.     

肾上腺髓质素真核表达载体对皮瓣缺血再灌注损伤的影响

背景：肾上腺髓质素可在心、脑、肾、肝等器官的缺血再灌注过程中起保护作用。目的：观察肾上腺髓质素真核表达载体对大鼠腹部皮瓣缺血再灌注损伤的影响及其作用机制。方法：构建大鼠肾上腺髓质素真核表达载体,将SD大鼠随机分假手术组、模型组、维拉帕米组、重组质粒组。重组质粒组腹部皮内注射肾上腺髓质素真核表达载体,其余各组注射等量生理盐水,各组注射4次后建立缺血再灌注模型,维拉帕米组于皮瓣内注射维拉帕米。结果与结论：与模型组相比,重组质粒组皮瓣组织中丙二醛、血管内皮素1的含量分别降低了38.50%（P〈0.01）,54.73%（P〈0.01）,超氧化物歧化酶的含量增加了50.67%（P〈0.05）。皮瓣组织学检查结果显示重组质粒组皮瓣炎性细胞浸润与水肿程度也较模型组明显减轻。结果证实肾上腺髓质素真核表达载体对大鼠皮瓣缺血再灌注损伤具有保护作用,其机制与减轻脂质过氧化及减少炎症细胞浸润有关。     

Evaluation of unfractionated heparin and recombinant hirudin on survival in a sustained ovine endotoxin shock model

OBJECTIVE: To compare and evaluate the potential benefit of two different anticoagulation regimens during endotoxemia in an ovine model. DESIGN: Animal prospective randomized and controlled study following preliminary dose-range study conforming with the Guide for the Care and Use of Laboratory Animals as promulgated by the Council of the American Physiologic Society and reviewed by the Ethical Committee for Animal Research of our institution. SETTING: Laboratories of anesthesiological investigations and hemostasis, primary care university medical center. SUBJECTS: Twenty-two adult sheep of either sex, weighing 29-42 kg (mean 35.8 kg), surgically instrumented for chronic studies and randomly allocated to receive three different treatment groups: unfractionated heparin (40 units.kg.hr; n = 7), recombinant hirudin (500 units.kg.hr; n = 7), or saline (nonanticoagulated controls; n = 8). INTERVENTIONS: Ovine model of severe endotoxin shock induced by a continuous intravenous endotoxin infusion over 72 hrs (10 ng.kg.min ). MEASUREMENTS AND MAIN RESULTS: Endotoxin infusion alone produced a progressive hypotensive, hyperdynamic shock state with right ventricle failure leading to the death of all animals of the control group within 44 hrs (median, 12 hrs). Heparin profoundly improved survival rate in this model, whereas effective anticoagulation with hirudin did not significantly increase the survival rate compared with controls. Animals in the control group died from disseminated intravascular coagulation, metabolic acidosis, and hemorrhagic pulmonary edema, whereas sheep treated with hirudin died from severe pulmonary edema (hypoxemia, increased alveoloarterial oxygen gradient associated with an increased wet/dry lung weight ratio) in the absence of disseminated intravascular coagulation. CONCLUSIONS: We conclude that systemic anticoagulation with the thrombin inhibitor hirudin is without benefit in this sheep model of lethal endotoxemia, whereas anticoagulation with heparin affords not only effective prevention of endotoxin-induced coagulation disorders but also global protection with prevention of respiratory decompensation and thus markedly improves survival rate in this situation.     

重组水蛭素克隆的表达及其产物的分离纯化

目的：在原核细胞中表达具有生物活性的重组水蛭素变异物１（ｒＨＶ１）并进行重组产物的分离纯化研究。方法：以质粒ｐＢＶ２２０为载体，在大肠杆菌ＤＨ５α中表达ｒＨＶ１，发色底物法测定其抗凝血酶生物活性；利用超滤、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０以及凝血酶亲合层析进行重组产物的分离纯化。结果：在大肠杆菌中获得了ｒＨＶ１的活性表达，表达量约占总蛋白的１６．９％，活力达到２０～３０ＡＴＵ／ｍｌ培养液；初步的纯化研究得到了具抗凝生物活性的ｒＨＶ１纯品，ＳＤＳ聚丙烯酰胺凝胶电泳呈单一条带。结论：重组水蛭素变异物１的活性表达及其分离纯化研究填补了国内空白，为研制国产高效重组抗凝药物带来希望。     

The effects of montelukast on random pattern skin flap survival: An experimental study in rats

Background: A variety of methods to improve skin flap survival, including the use of pharmacologic agents, have been intensively investigated. Decreasing neutrophil-mediated inflammation and tissue injury has been reported to be effective in improving flap survival. Montelukast is a selective reversible cysteinyl leukotriene receptor-1 antagonist that has been found to have protective effects against renal ischemia reperfusion injury and burn-induced oxidative injury of the skin in rats. However, its effects on skin flap survival have not been previously reported.Objective: The aim of this study was to investigate the effects of montelukast on neutrophil-mediated random pattern skin flap survival.Methods: Male Sprague-Dawley rats weighing 230 to 250 g were randomly divided into 2 groups—the montelukast-treated group and the control group. Caudally based rectangular random pattern skin flaps 3 × 9 cm were elevated on the backs of the rats. The flaps were sutured into their original places. In the montelukast group, 1 mL of solution containing 10 mg/kg montelukast was administered intraperitoneally (IP) 30 minutes before surgery and then daily for 6 days. In the control group, 1 mL of saline was administered IP 30 minutes before surgery and then daily for 6 days. To observe the effects of montelukast, myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration, was measured from extracted skin tissue 12 hours after flap elevation. Flap viability was evaluated 7 days after surgery by measuring necrotic flap area and total flap area.Results: Sixteen rats (mean [SD] weight, 240.6 [6.6] g) were equally divided between the 2 groups. All rats survived throughout the study period. Mean (SD) MPO activity in flap tissue was significantly lower in the montelukast group than in the control group (14.57 [2.33] vs 21.28 [4.86] U/g protein; P = 0.005). The percentage of necrotic flap area was significantly lower in the montelukast group than in the control group (17.17 [7.95] vs 37.51 [10.72]; P = 0.001).Conclusion: This small, experimental, in vivo animal study found that montelukast was associated with both lower MPO activity and a lower percentage of necrotic random pattern skin flap area. Future studies are needed to clarify these findings.     

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity – a staphylokinase variant. I. In vitro study

We attempted to construct a new recombinant protein characterized by fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. To the C-terminal part of recombinant staphylokinase (r-SAK), which is a promising profibrinolytic agent, we assembled: (i) the Kringle 2 domain (K2) of tissue-type plasminogen activator (t-PA), containing a fibrin-specific binding site, (ii) the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation and (iii) the antithrombotic agent - hirudin. The cDNA for hybrid protein SAK-RGD-K2-Hir was cloned into pESP-3 yeast protein expression vector. The introduction of K2 t-PA, RGD sequence and hirudin into r-SAK molecule did not alter the SAK activity.The plasminogen activation rate (determined by K(M) and K(cat)) of SAK-RGD-K2-Hir was not significantly different from that of r-SAK. Affinity and binding strength of the recombinant protein to fibrin immobilized on the biosensor were higher than to r-SAK. We observed a higher clot lysis potency of SAK-RGD-K2-Hir as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirudin part of the recombinant fusion protein SAK-RGD-K2-Hir was the same as that of r-Hir alone. In conclusion, the results of the in vitro study suggest that the SAK-RGD-K2-Hir construct can be a more potent and faster-acting thrombolytic agent with antithrombin and antiplatelet properties compared with standard r-SAK.     

皮瓣移植及免疫调节

背景：皮肤损伤非常常见,皮瓣移植是治疗皮肤损伤的方法之一,而且已被广泛应用。设计合理的皮瓣移植,抑制移植后排斥反应,可以改善移植皮瓣的存活状态。目的：研究皮瓣移植后的免疫调节机制及抑制调节因素,为皮瓣移植的免疫调节研究提供参考信息。方法：检索万方数据库2002至2011年收录皮瓣移植后免疫调节相关研究文献,并进行深入分析,对皮瓣移植动物模型建立可行性的实验研究和一氧化氮、成纤维细胞生长因子、表皮生长因子对皮瓣移植的影响以及皮瓣移植后的微循环保护研究和感觉神经末梢再生研究进行实验数据分析,以明确影响移植皮瓣存活的因素,并进行调控,延长移植皮瓣的存活期,改善皮瓣移植后的存活状态。结果与结论：皮瓣移植的大鼠实验模型是一种很好的皮瓣移植动物模型,可以进行各种移植皮瓣的模型建立,并且可以用于皮瓣移植免疫以及微循环等各方面的实验研究。在皮瓣移植后的免疫调节中,一氧化氮和成纤维细胞生长因子以及表皮生长因子均可以对免疫反应进行调控影响,而异丙酚以及盐酸罂粟碱等物质可以对皮瓣移植后的微循环保护起到一定的作用。