富血小板血浆凝胶制备及对大鼠皮瓣成活的作用分析

目的寻找制备富血小板血浆(platelet-rich plasma,PRP)的最佳离心方案,并制备PRP凝胶用于皮下注射促进大鼠皮瓣成活,探讨其有效性及作用机制。方法取12周龄健康Wistar大鼠72只,体重250～300 g。于24只大鼠心脏取动脉血8～10 mL/只,分别采用3种离心方法制备PRP。A组:以200×g离心15 min、500×g离心10 min;B组:以312×g离心10 min、1 248×g离心10 min;C组:以200×g离心15 min,200×g离心10 min。在PRP制作过程中取各组全血、PRP及贫血小板血浆(platelet-poor plasma,PPP)行血小板计数,根据血小板计数结果选择最佳离心方案,并取对应的PRP、PPP及第1次离心后血清,采用ELISA法测定PDGF-BB和TGF-β1浓度;并制备PRP及PPP凝胶。于48只大鼠背部制备大小为11 cm×3 cm的皮瓣,随机分为3组(n=16):PRP组每只大鼠皮瓣下注射100 mL PRP凝胶,PPP组同法注射100 mL PPP凝胶,对照组不作处理。术后大体观察皮瓣成活情况,7 d后取材计算成活率;组织学观察计数炎性细胞,免疫组织化学染色计数微血管;于术后8、24 h,3、7 d行实时荧光定量PCR检测VEGF、EGF、PDGF-AA和PDGF-BB mRNA表达。结果血小板计数结果示,A组血小板浓缩倍数最高,为最佳制备方法。A组PRP中TGF-β1、PDGF-BB浓度明显高于血清及PPP(P<0.05)。与对照组相比,术后PRP组伤口无脓性分泌物;PRP组皮瓣成活率为61.2%±9.1%,与PPP组35.8%±11.3%及对照组28.0%±5.4%比较,差异有统计学意义(P<0.05)。PRP组炎性细胞计数较PPP组及对照组明显减少,微血管计数明显高于其余两组,差异均有统计学意义(P<0.05)。实时荧光定量PCR检测示与PPP组及对照组比较,PRP组VEGF及PDGF-BB mRNA术后均高表达,而EGF mRNA仅在术后24 h内呈高表达,PDGF-AA mRNA在3 d后开始高表达。术后3 d及7 d PRP组PDGF-AA、8 h PDGF-BB、24 h及3 d VEGF、24 h EGF的mRNA相对表达量与其他两组比较,差异有统计学意义(P<0.05)。结论以200×g离心15 min、500×g离心10 min是制备PRP的最佳离心方案。PRP凝胶可通过调节血管生发相关基因促进大鼠皮瓣成活。

Preparation of platelet-rich plasma gel and its effect on skin flap survival of rat

Objective To find out the best method to prepare platelet-rich plasma(PRP) and to evaluate the effect of PRP gel on skin flap survival and its mechanism.Methods Totally,72 Wistar rats(aged 12 weeks,weighing 250-300 g) were used for the experiment.The arterial blood(8-10 mL) were collected from the hearts of 24 rats to prepare PRP with three kinds of centrifuge methods: in group A,200 × g centrifuge for 15 minutes,and 500 × g centrifuge for 10 minutes;in group B,312 × g centrifuge for 10 minutes,and 1 248 × g centrifuge for 10 minutes;and in group C,200 × g centrifuge for 15 minutes,and 200× g centrifuge for 10 minutes.The platelet was counted in the whole blood,PRP,and platelet-poor plasma(PPP) to determine an ideal centrifuge.PRP,PPP,and the serum after first centrifuge were collected.The concentrations of platelet-derived growth factor BB(PDGF-BB) and transforming growth factor β1(TGF-β1) were measured in the PRP,PPP,and serum using the enzyme-linked immunosorbent assay method,and PRP and PPP gels were prepared.The flaps of 11 cm × 3 cm in size were elevated on the back of 48 rats,which were divided into 3 groups: PRP gel(PRP group,n=16) and PPP gel(PPP group,n=16) were injected,no treatment was given in the control group(n=16).The flap survival rate was measured at 7 days.Histological and real-time PCR were used to count the inflammatory cells and blood vessel density,and to detect the expressions of vascular endothelial growth factor(VEGF),epidermal growth factor(EGF),PDGF-AA,and PDGF-BB mRNA at 8 hours,24 hours,3 days,and 7 days.Results Platelet counting showed platelet in group A was the highest.ELISA evaluation showed that the concentrations of TGF-β1 and PDGF-BB were significantly higher in PRP than in PPP and serum(P < 0.05).The flap survival rate was 61.2% ± 9.1% in PRP group,showing significant differences(P < 0.05) when compared with that in PPP group(35.8% ± 11.3%) and control group(28.0% ± 5.4%).The inflammatory cells were significantly lower and the blood vessel density was significantly higher in PRP group than in PPP group and control group(P < 0.05).When compared with PPP group and control group,the expressions of VEGF and PDGF-BB increased at all time after operation in PRP group;the expression of EGF increased within 24 hours;and the expression of PDGF-AA increased after 3 days.There were significant differences in PDGF-AA mRNA at 3 days and 7 days,PDGF-BB mRNA at 8 hours,VEGF mRNA at 24 hours and 3 days,and EGF mRNA at 24 hours between PRP group and PPP and control groups(P < 0.05).Conclusion 200 × g centrifuge for 15 minutes and 500×g centrifuge for 10 minutes is the best PRP preparation method.PRP can improve the skin flap survival by regulating the genes involved in angiogenesis.