局部应用重组水蛭素对兔耳静脉淤血皮瓣存活的影响

The Effect of the Topical Application of Recombinant Hirudin on the Survival of Rabbit Ear Flap of Venostasis

目的探讨局部应用水蛭素对静脉淤血皮瓣成活的影响及其作用机制。方法将18只健康大耳白兔随机分为3组:1个对照组(A组:注射生理盐水组)和2个治疗组(B组:低分子肝素治疗组;C组:重组水蛭素治疗组),每组6只。在左耳背部设计3 cm×6 cm的含耳中央血管、神经为蒂(蒂宽1cm)的轴型皮瓣,于蒂部结扎并切断耳中央静脉和神经,并结扎其他旁支静脉,创面用5—0丝线结扎止血,制成以耳中央动脉为唯一血供,1cm宽的蒂部为唯一静脉回流途径的静脉淤血皮瓣模型。治疗组在术后立即分别给予皮瓣下多点、均匀注射水蛭素(1U/1ml/皮瓣)或低分子肝素(0.5mg/1ml/皮瓣),对照组同法给予等量(1ml)生理盐水,皮瓣用5—0丝线原位缝合。术后观察皮瓣表面毛发脱落、血肿形成情况和皮瓣存活率,利用组织学观察毛细血管血栓形成情况以及中性粒细胞附壁及红细胞聚集情况和免疫组化检测皮瓣内VEGF蛋白的含量。结果①肉眼观察:皮瓣形成后所有皮瓣均肿胀明显,远端淤血形成,颜色暗红,对照组颜色明显深于治疗组,水蛭素治疗组1例(1/6),肝素治疗组2例(2/6),生理盐水对照组4例(4/6)出现明显的血肿。②各组皮瓣存活率:低分子肝素及重组水蛭素治疗组成活面积百分比分别为(92.33±1.67)%与(94.83±1.91)%,均比生理盐水对照组的(77.93±1.18)%高,有显著差异(P<0.01),重组水蛭素治疗组与低分子肝素治疗组的皮瓣存活率无显著统计学差异(P>0.05)。③组织学及免疫组化:治疗组皮下毛细血管内血栓形成及红细胞聚集情况均比对照组轻,重组水蛭素治疗组皮瓣中VEGF表达较其他两组增多。结论重组水蛭素的应用可明显改善皮瓣的静脉淤血,促进皮瓣血管生成,提高皮瓣的成活率,并可避免发生肝素应用过程中的血小板减少症等严重并发症,可代替肝素在临床上的应用。

Objective To evaluate the effect and mechanism of topical application of hirudin on the survival of flap with passive congestion. Methods 18 healthy big ear white rabbits were randomly divided into 3 groups, a control group （ Group A ： saline injection group） and two treatment groups （ Group B ： low - molecular - weight heparin injection group, Group C ： recombinant hirudin injection group）, 6 rabbits each group. An axial flap based on ear＇s central blood vessels and nerves （base width 1cm） was designed in size of 3 ×6cm on the back of the left ear with the ligation and cutting off of ear＇s central vein and nerves, other branch veins were also ligated, the wound was sutured with 5 -0 silk to stop bleeding, thus the model of flap with passive congestion was set up to be with the only blood supply of ear central artery and the only venous return of the lcm - wide base. The flaps in treatment groups were given immediately after operation subcutaneous injection of multipoint well - distributed hirudin （1U/1ml/flap） or low - molecular - weight heparin （0. 5mg/1ml/flap） respectively, equivalent amount of saline （ 1 ml） was given to the control group in the same way, and the wound was the su- tured with 5 - 0 silk in situ. The status of hair loss and hematoma on the flap and the survival rate of the flap were observed postoperatively. Histological observation of the capillary thrombosis, the wall attachment of neutrophil and the erythrocyte aggregation and immunohistoehemical analysis of content of VEGF protein in flap were also conducted. Results （ 1 ） Visual observation： All flap appeared significant swell with congestion at the far end in the color of dark red. The colors of the flaps in the control group were significantly more heavy than that of the treatment groups. Obvious hematoma appeared in different number of cases in hirudin injection group （1/6）, heparin injection group （2/6） and saline control group （4/6） . （ 2 ） The survival rate of flaps ： the survival area of low - molecular - weight heparin injection group and recombinant hirudin injection group were 92. 33 ± 1.67% and 94. 83 ±1.91 respectively, both higher than that of saline control group （77.93 ± 1.18% ） with significant difference （P 〈0. 01 ） . There was no significant difference between the two treatment groups （ P 〉 0. 05 ） . （3） Histology and immunohistochemistry ： The thrombi forming in subcutaneous capillaries and the erythrocyte aggregation in treatment groups were less than that in control group. Expression of VEGF in recombinant hirudin injection group was more than the other groups. Conclusion Application of the recombinant hirudin can significantly alleviate the venous passive congestion in the flaps, promote angiogenesis, and improve the survival rate of the flap. In addition, it can avoid severe complications like thrombocytopenia happened in the application of heparin. The recombinant hirudin can be used clinically instead of heparin.