纳米银-猪小肠黏膜下层治疗肛瘘的愈合机制  

Healing mechanism of porcine small intestinal submucosa decorated with nano-silver in the teatment of anal fistula

作　　者：陶禹 李干斌 张皓宇 曹可 王振军[2] 韩加刚[2] Tao Yu;Li Ganbin;Zhang Haoyu;Cao Ke;Wang Zhenjun;Han Jiagang(Department of Colorectal Surgery,Second Affiliated Hospital of Naval Medical University,Shanghai 200003,China;Department of General Surgery,Beijing Chaoyang Hospital Affiliated to Capital Medical University,Beijing 100020,China)

机构地区：[1]海军军医大学第二附属医院肛肠外科,上海200003 [2]首都医科大学附属北京朝阳医院普通外科,100020

出　　处：《中华实验外科杂志》2022年第8期1516-1522,共7页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82070685);首都卫生发展科研专项重点攻关(2018-1-2032);北京朝阳医院1351人才培养计划项目(CYXZ-2017-09);北京市属医院科研培育项目(PX2019012);首都医科大学科研培养基金(PYZ2018089)。

摘　　要：目的探讨纳米银-猪小肠黏膜下层(NS-PSIS)治疗肛瘘动物模型的愈合机制。方法建立新西兰兔肛瘘动物模型, 根据随机数字表随机分为实验组和对照组各12只。实验组使用NS-PSIS填塞治疗肛瘘, 对照组使用猪小肠黏膜下层(PSIS), 并在术后12、24、48 h及14 d获取肛瘘组织标本(每组3只)。行苏木素-伊红染色检查肛瘘组织的愈合情况。行Masson染色评估肛瘘边缘组织中胶原沉积情况, 并定量分析。行免疫组织化学方法检测肛瘘边缘组织中CD34的表达, 计算微血管密度(MVD);检测转化生长因子-β1(TGF-β1)、Smad2、COLⅠ和COLⅢ的蛋白表达, 并定量分析。行实时荧光定量聚合酶链式反应(RT-qPCR)检测肛瘘边缘组织中TGF-β1/Smad2/COLⅠ/COLⅢ信号通路相关分子TGF-β1、Smad2、COLⅠ和COLⅢ的mRNA表达。符合正态分布的计量资料, 两组间比较采用独立样本t检验, 否则采用Wilcoxon秩和检验。结果成功构建NS-PSIS, 并建立新西兰兔肛瘘模型。NS-PSIS和PSIS填塞治疗肛瘘早期(术后12~48 h), 两组的瘘道周围均可见明显炎性细胞浸润、成纤维细胞增殖、胶原合成和新生血管形成。Masson染色显示, 在术后48 h, NS-PSIS组的胶原合成明显多于PSIS组[(29.40±2.80)%比(20.97±1.68)%, t=-7.752, P<0.01]。免疫组织化学显示, 在术后48 h, NS-PSIS组的MVD明显高于PSIS组[6.8(5.2, 7.8)比5(4, 6.8), Z=-2.969, P<0.01];在术后24 h, NS-PSIS组COLⅠ[(22.06±2.83)%比(13.5±2.88)%, t=-6.342, P<0.01]和COLⅢ[(25.70±3.44)%比(20.02±2.45)%, t=-4.038, P<0.01]的表达水平均明显高于PSIS组;在术后48 h, NS-PSIS组的COLⅠ[(28.71±3.81)%比(20.09±3.07)%, t=-5.284, P<0.01]和COLⅢ[(30.59±1.41)%比(24.01±1.77)%, t=-8.710, P<0.01]的表达水平均明显高于PSIS组;此外, 在术后48 h, NS-PSIS组的TGF-β1[19.71(18.56, 20.90)%比13.50(11.34, 14.71)%, Z=-3.621, P<0.01]和Smad2[(18.56±2.57)%比(12.22±2.55)%, t=-5.249, P<0.01]的表达水平明显高于PSIS组。RT-qPCR�Objective To explore the healing mechanism of porcine small intestinal submucosa decorated with nano-silver(NS-PSIS)in the treatment of a model of fistula-in-ano.Methods NS-PSIS was constructed.Animal models of fistula-in-ano were established using New Zealand rabbits and randomly divided into two groups according to random number table(n=12 in each group).Rabbits in the experimental group were treated with NS-PSIS for anal fistulas,and those in the control group were treated with PSIS.The treated anal fistula tissue samples were obtained at 12,24,48 h and 14 d after surgery(n=3 in each group).Hematoxylin-eosin staining was performed to observe the pathomorphological changes of the marginal tissues of the anal fistula.Masson staining was performed to assess collagen deposition in the marginal tissue of the anal fistula and quantitative analysis was performed.Immunohistochemical staining was performed to detect the expression of CD34 in the marginal tissue of the anal fistula and calculate the microvessel density(MVD).The expression of transforming growth factor(TGF)-β1,Smad2,collagen typeⅠ(COLⅠ)and collagen typeⅢ(COLⅢ)was detected.Real-time polymerase chain reaction(RT-qPCR)was performed to detect the mRNA expression of TGF-β1,Smad2,COLⅠand COLⅢ.For measurement data meeting normal distribution,independent sample t-test was used for comparison between two groups;otherwise,Wilcoxon rank sum test was used.Results NS-PSIS was successfully constructed.A New Zealand rabbit model of fistula-in-ano was successfully established.In the early stage of treatment(12-48 h after surgery),significant inflammatory cell infiltration,fibroblast proliferation,collagen synthesis and neovascularization were observed around the fistula tract in both groups.Masson staining showed that collagen synthesis in the NS-PSIS group was significantly more than that in the PSIS group at 48 h after surgery[(29.40±2.80)%vs.(20.97±1.68)%,t=-7.752,P<0.01].Immunohistochemistry showed that at 48 h after surgery,the MVD in NS-PSIS group

关 键 词：肛瘘 猪小肠黏膜下层 纳米银 动物模型 愈合 

分 类 号：R657.16[医药卫生—外科学]