摘要目的 探讨自体富血小板血浆(PRP)对游离皮瓣修复兔软组织缺损的影响. 方法 取30只6个月龄、雌雄不拘新西兰大白兔,心脏采血后Aghaloo法制备PRP,于每只兔耳制作5 cm×3 cm的游离皮瓣模型.采用随机数字表法选择每只兔一侧兔耳作为PRP组,另一侧兔耳作为生理盐水组.PRP组兔耳皮瓣基底均匀涂抹1.0 mL自体PRP,生理盐水组涂抹等量生理盐水,皮瓣原位回植.分别于术后2、3、5、7、14 d,每组取6只兔,观察并记录兔耳皮瓣成活情况;取皮瓣组织,行苏木精-伊红染色观察皮瓣基底组织形态;采用免疫荧光法检测皮瓣基底组织的CD31和α平滑肌肌动蛋白(α-SMA)表达.另取1只同等实验条件下不制备皮瓣的6个月龄雄性新西兰大白兔,分别采集全血和制备PRP,行血小板计数及采用双抗体夹心酶联免疫吸附测定法检测血管内皮生长因子(VEGF)和转化生长因子β(TGF-β)含量.对数据行析因设计方差分析、配对样本t检验及Bonferroni校正. 结果 (1)术后2d,PRP组兔创面皮瓣颜色彤红,与基底组织贴合良好;生理盐水组兔创面皮瓣颜色暗红,与基底组织贴合不良.术后3d,PRP组兔创面皮瓣颜色红润,与基底组织贴合紧密;生理盐水组兔创面皮瓣散在斑状暗红,与基底组织贴合一般.术后5d,PRP组兔创面皮瓣淡红,与基底组织贴合紧密,皮瓣已成活;生理盐水组兔创面皮瓣与基底组织贴合紧密,皮瓣颜色红润.术后7d,PRP组兔创面皮瓣表面覆盖中量兔毛,皮瓣颜色与周围皮肤颜色接近;生理盐水组兔创面皮瓣与基底贴合一般,表面仅有少量绒毛覆盖.术后14 d,PRP组兔创面愈合良好,生理盐水组仍有小创面尚未愈合.(2)术后2d,2组兔创面皮瓣均有炎性细胞浸润.术后3d,PRP组兔创面皮瓣可见新生微血管,间质出血少;生理盐水组兔创面皮瓣新生微血管少.术后5d,生理盐水组兔创面皮瓣中量炎性细胞浸润,新生微血管数量少;PRP组兔创面皮瓣可见大量Fb,少量炎性细胞浸润,明显可见周围散布新生微血管.术后7d,生理盐水组兔创面皮瓣新生微血管数量较PRP组少.术后14 d,PRP组兔创面皮瓣新生微血管分化逐渐成熟,周围大量Fb分布;生理盐水组兔创面皮瓣部分新生微血管分化成熟,愈合过程较PRP组迟缓.(3)术后2、3、5、7、14 d,PRP组兔创面皮瓣基底组织的CD31和α-SMA表达量均明显多于生理盐水组(t=10.133、5.444、9.450、6.986、8.394,14.896、10.328、9.295、13.902、10.814,P＜0.01).(4)活化后的兔PRP中血小板计数为(2 863 ±962)×109/L,明显高于全血中的(393±49)×109/L(t=7.690,P＜0.05).(5)活化后的兔PRP中VEGF、TGF-β含量分别为(564.3±3.2)、(1 143 ±251)pg/mL,明显高于全血中的(99.7±0.4)、(274±95)pg/mL(t=287.390、9.648,P＜0.05或P＜0.01). 结论 兔PRP中含有大量的VEGF与TGF-β,可以有效促进游离皮瓣组织微血管新生,加快游离皮瓣成活.

abstractsObjective To explore the effects of autologous platelet-rich plasma (PRP) in the repair of soft tissue defects of rabbits with free flap.Methods Thirty 6-month-old New Zealand white rabbits,male and female unlimited,were used to harvest blood from the heart.PRP was prepared by Aghaloo method,then free flap model with size of 5 cm × 3 cm was reproduced on each ear of the rabbit.According to the random number table,one ear of each rabbit was recruited to PRP group,and the other ear was recruited to normal saline group.The base of flap on rabbit ear in PRP group was evenly spread with 1.0 mL autologous PRP,and equivalent volume of normal saline was applied to that in normal saline group.Then,the flap was replanted in situ.On post surgery day (PSD) 2,3,5,7,and 14,6 rabbits in each group were taken.The survival of flap was observed and recorded.The morphology of the basal tissue of flap was observed by hematoxylin-eosin staining.The expressions of CD31 and α smooth muscle actin (α-SMA) in the basal tissue of flap were detected by immunofluorescence method.Another 6-month-old male New Zealand white rabbit without making flap under the same experimental conditions was used for harvesting whole blood and preparing PRP.Then blood platelet count in whole blood and PRP was determined,and the content of vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) was detected by double-antibody sandwich enzyme-linked immunosorbent assay.Data were processed with analysis of variance of factorial design,paired sample t test,and Bonferroni correction.Results (1) On PSD 2,the flaps of wounds of rabbits in PRP group were reddish and adhered well to the basal tissue;the flaps of wounds of rabbits in normal saline group were dark red and poorly attached to the basal tissue.On PSD 3,the flaps of wounds of rabbits in PRP group were ruddy and closely adhered to the basal tissue;the flaps of wounds of rabbits in normal saline group were scattered in the plaque-like dark red and generally attached to the base.On PSD 5,the flaps of wounds of rabbits in PRP group were reddish and closely adhered to the basal tissue,and the flaps were alive;while flaps of wounds of rabbits in normal saline group were rosy and closely adhered to the basal tissue.On PSD 7,the surface of flaps of wounds of rabbits in PRP group was covered with a medium amount of rabbit hair.The color of flap was similar to that of the surrounding skin.The flaps of wounds of rabbits in normal saline group were generally attached to the base,and the surface was only covered with a small amount of fluff.On PSD 14,the incisions were healed well in PRP group,while small wounds in normal saline group were not healed.(2) On PSD 2,inflammatory cell infiltration was observed in flaps of wounds of rabbits in both groups.On PSD 3,the flaps of wounds of rabbits in PRP group showed neovascularization,with less interstitial hemorrhage;while there were less neovascularization in the flaps of wounds of rabbits in normal saline group.On PSD 5,a medium number of inflammatory cell infiltration and a small amount of new microvessels were observed in flaps of wounds of rabbits in normal saline group.Many fibroblasts,a small amount of inflammatory cells,and scattered new microvessels were observed in flaps of wounds of rabbits in PRP group.On PSD 7,the number of new microvessels in normal saline group was significantly lower than that in PRP group.On PSD 14,the new microvessels in the flaps of wounds of rabbits in PRP group gradually matured,and a large number of fibroblasts distributed around them.Some of the newly formed microvessels in the flaps of wounds of rabbits in normal saline group were mature,and the healing was slower than that of PRP group.(3) On PSD 2,3,5,7,and 14,the expressions of CD31 and α-SMA in the basal tissue of flaps of wounds of rabbits in PRP group were significantly higher than those in normal saline group (t =10.133,5.444,9.450,6.986,8.394,14.896,10.328,9.295,13.902,10.814,P ＜ 0.01).(4) The platelet count in activated PRP of rabbits was (2 863 ± 962) × 109/L,which was significantly higher than (393 ± 49) × 109/L in whole blood (t =7.690,P ＜ 0.05).(5) The content of VEGF and TGF-β in activated PRP of rabbits was (564.3 ± 3.2) and (1 143 ± 251) pg/mL,which was significantly higher than (99.7 ± 0.4) and (274 ± 95) pg/mL in whole blood,respectively (t =287.390,9.648,P ＜ 0.05 or P ＜ 0.01).Conclusions PRP of rabbits contains high concentrations of VEGF and TGF-β.Therefore,PRP can effectively promote microvascular regeneration in free flap tissue and accelerate the survival of free flap.