自体浓缩生长因子凝胶治疗肛瘘的疗效及作用机制研究  

Application of concentrated autologous growth factor-containing gel for treatment of anal fistula in pig model

作　　者：张秀峰[1] 王厚东[1] 裘建明[1] 鲁振锋 邵书先[1] 沈忠[1] ZHANG Xiufeng;WANG Houdong;QIU Jianming;LU Zhenfeng;SHAO Shuxian;SHEN Zhong(Department of Coloproctology,Hangzhou Third People's Hospital,Hangzhou 310009,China)

机构地区：[1]杭州市第三人民医院肛肠科,310009

出　　处：《浙江医学》2023年第15期1603-1608,1620,I0003,共8页Zhejiang Medical Journal

基　　金：浙江省公益技术应用研究资助项目(LGF20H030002);杭州市卫生计生科技计划项目(2017A26、A20210250);杭州市科技计划引导项目(20171226Y17)。

摘　　要：目的探讨自体浓缩生长因子(CGF)凝胶治疗肛瘘的疗效及作用机制。方法采用挂线法对6只猪进行肛瘘造模,30 d后行瘘管造影及病理检查确认造模成功。将左右侧瘘管按随机数字表法分为凝胶组(填塞CGF凝胶)和对照组;观察两组瘘管创面愈合情况,检测术后第7天瘘管外口组织样本血小板衍生生长因子(PDGF)、增殖细胞核抗原(PCNA)、α平滑肌肌动蛋白(α-SMA)、c-fos mRNA及蛋白表达水平,以及细胞外调节蛋白激酶(ERK)信号通路因子[包括丝裂原激活蛋白激酶(MEK)1/2、磷酸化MEK(p-MEK)1/2、ERK1/2、磷酸化ERK(p-ERK)1/2]蛋白表达水平。取人皮肤成纤维细胞(HSF),按随机数字表法分为对照组(10%FBS)、20%CGF组、20%CGF+干扰RNA(siRNA)组、20%CGF+siRNA ERK组,采用免疫荧光法检测CGF对HSF细胞增殖能力的影响。结果动物实验结果显示,凝胶组创面愈合时间明显短于对照组(P<0.05),瘘管外口组织样本PDGF、PCNA、α-SMA、c-fos mRNA及蛋白表达水平均明显高于对照组(均P<0.05),p-MEK1/2、p-ERK1/2蛋白水平均明显高于对照组(均P<0.05),MEK1/2、ERK1/2蛋白表达水平比较差异均无统计学意义(均P>0.05)。细胞实验结果显示,与对照组比较,20%CGF组、20%CGF+siRNA组HSF细胞增殖百分比均明显升高(均P<0.05);与20%CGF+siRNA组比较,20%CGF+siRNA ERK组HSF细胞增殖百分比明显降低(P<0.05)。结论CGF凝胶治疗肛瘘有效,其作用机制是通过上调ERK信号通路因子水平来促进HSF增殖,从而加速瘘管愈合。Objective To investigate the effect and mechanism of autologous concentrated growth factor(CGF)gel in treating anal fistula.Methods Anorectal fistulas were modeled in 6 pigs by hanging line method.The anal fistula model was verified by fistulography and histopathological evaluation 30 d after surgery.Left and right fistula were randomly selected as CGF group(full filled with CGF gel)and the control group.The wound healing of fistulas in the two groups was observed,and the mRNA and protein expression levels of platelet-derived growth factor(PDGF),proliferating cell nuclear antigen(PCNA),alpha smooth muscle actin(α-SMA),c-fos and the protein expression levels of extracellular regulated protein kinase(ERK)signaling pathway factors,including mitogen activated protein kinase(MEK)1/2,p-MEK1/2,ERK1/2,p-ERK1/2,were detected in fistulas external opening tissue samples on the 7th day after surgery.Human skin fibroblast(HSF)was divided into control group(10%FBS),20%CGF group,20%CGF+small interfering RNA(siRNA)group and 20%CGF+siRNA ERK group by random number table method.The effects of CGF on the proliferation of HSF cells were detected by immunofluorescence method.Results The results of animal experiments showed that the wound healing time in the gel group was significantly shorter than that in the control group(P<0.05),and the mRNA and protein expression levels of PDGF,PCNA,α-SMA and c-fos in fistula external opening tissue samples were significantly higher than those in the control group(all P<0.05).The protein levels of p-MEK1/2 and p-ERK1/2 were significantly higher than those of control group(all P<0.05),but there were no significant differences in the protein expression levels of MEK1/2 and ERK1/2(all P>0.05).The cell experiment results showed that compared with the control group,the proliferation percentage of HSF in 20%CGF group and 20%CGF+siRNA group were significantly increased(both P<0.05).Compared with 20%CGF+siRNA group,the proliferation percentage of HSF in 20%CGF+siRNA ERK group was significantly decreased

关 键 词：自体浓缩生长因子 肛瘘 人皮肤成纤维细胞 血小板衍生生长因子 

分 类 号：R657.16[医药卫生—外科学]