自体富血小板血浆用于肛瘘术后创面修复的实验研究

The study of autologous platelet-rich plasma in the repair of perianal tissue defects following anal fistulectomy

目的 探索自体富血小板血浆(PRP)修复肛瘘术后创面的作用。方法 10只实验用猪采用挂线法进行肛瘘造模。38 d后瘘管造影及病理检查评估瘘管形成造模成功。瘘管切除术后左、右侧肛周创面随机数字表法分为PRP组与乏血小板血浆(PPP)组,PRP组创面予以PRP注射,PPP组创面予以PPP注射。术后第12天取创面中央组织样本,酶联免疫吸附试验(ELISA)检测组织样本中表皮细胞生长因子(EGF)和促血管生成素-2(Ang-2)含量,免疫组化检测CD34表达水平,免疫荧光检测波形蛋白(Vimentin)表达情况。ELISA法检测炎症细胞因子白介素-6(IL-6)及白介素-12(IL-12)含量。观察创面愈合时间,超声评估创面愈合效果。结果 PRP中血小板浓度为(668.90±188.30)×109/L,显著高于外周静脉血(194.10±44.44)×109/L及PPP(80.90±28.58)×109/L(F=76.320,P<0.01)。ELISA检测结果显示,PRP注射组创面组织中EGF及Ang-2含量均显著高于PPP注射组[(293.72±21.21)pg/mL比(243.94±38.14)pg/mL,t=4.839,P<0.01;(87.29±9.46)pg/mL比(71.10±8.06)pg/mL,t=5.527,P<0.01]。免疫组化显示,PRP注射组的CD34水平显著高于PPP注射组(0.20±0.03比0.15±0.03,t=3.181,P=0.01),免疫荧光检测结果显示,Vimentin蛋白在PRP注射组的组织中的水平高于PPP注射组(61.30±6.57比27.49±3.77,t=7.731,P<0.01)。ELISA检测显示,炎症因子IL-6及IL-12在PPP注射组的创面组织中含量均显著高于PRP注射组[(639.53±70.34)pg/mL比(469.91±51.16)pg/mL,t=8.274,P<0.01;(194.84±21.81)pg/mL比(156.84±18.52)pg/mL,t=5.633,P<0.01]。PRP组愈合时间为(16.9±2.0)d,明显低于PPP组(21.2±2.1)d(t=-4.660,P<0.01)。超声检查结果显示,两组未均出现假性愈合。结论 PRP局部注射肛瘘术后创面可以提高创面组织中EGF及Ang-2含量,从而促进上皮生长及新生血管形成,同时可以减轻组织炎症反应,促进肛瘘术后创面愈合。

Objective To evaluate the effect of autologous platelet-rich plasma(PRP)for the repair of perianal tissue defect following anal fistulectomy.Methods Ten healthy pigs underwent rubber band ligation surgery,and fistulography and histopathological analysis were performed to assess the success of fistula formation modeling on the 38th day after surgery.After fistulectomy,left and right perianal defects were randomly divided into two groups:the experimental group received PRP injection,and the control group received platelet-poor plasma(PPP)injection.Tissue samples were taken from the central of wounds on day 12 after the surgery.The levels of epidermal growth factor(EGF)and angiopoietin-2(Ang-2)in wound tissues were measured by enzyme linked immunosorbent assay(ELISA).At the same time,immunohistochemical analysis of CD34 expression and immunofluorescence assay of Vimentin expression in wound tissues were carried out.The expression levels of inflammatory cytokine interleukin-6(IL-6)and IL-12 in wound tissues were also measured by ELISA.Wound healing times were monitored,and ultrasound was used to evaluate the tissue healing situations.Results The platelet count was significantly higher in PRP group than in peripheral venous blood and PPP group[(668.90±188.30)×109/L vs.(194.10±44.44)×109/L and(80.90±28.58)×109/L;F=76.342,P<0.01].The results of ELISA showed that the levels of EGF expression and Ang-2 expression were significantly higher in the PRP group than in the PPP group(EGF:293.72±21.21 pg/mL vs.243.94±38.14 pg/mL,t=4.839,P<0.01;Ang-2:87.29±9.46 pg/mL vs.71.10±8.06 pg/mL,t=5.527,P<0.01).Immunohistochemical analysis revealed that the CD34 level in PRP group was significantly higher than that in PPP group(0.20±0.03 vs.0.15±0.03,t=3.181,P=0.01).Vimentin expression as measured by immunofluorescence assay was higher in PRP group than in PPP group(61.30±6.57 vs.27.49±3.77,t=7.731,P<0.01).ELISA also revealed that the levels of IL-6 and IL-12 expression in PPP group were significantly higher than those in the PRP group(IL-6:639.53±70.34 pg/mL vs.469.91±51.16 pg/mL,t=8.274,P<0.01;IL-12:194.84±21.81 pg/mL vs.156.84±18.52 pg/mL,t=5.633,P<0.01).The PRP group experienced significantly faster wound healing than the control group(16.9±2.0 d vs.21.2±2.1 d,t=-4.664,P<0.01).The ultrasound scan revealed that neither group exhibited pseudohealing.Conclusion After anal fistulectomy,local injection of PRP could reduce the inflammatory response and increase the level of EGF and Ang-2 in the wound tissue,thereby promoting epithelial growth and neovascuarization and accelerating wound healing.