基于TGF-β1/Smads/ACTA2信号通路探讨猪食管ESD环切术后狭窄模型人工溃疡纤维化的机制

The mechanism of artificial ulcer fibrosis of stenosis after full circumcision of esophageal mucosal dissection based on TGF-β1/Smads/ACTA2 signaling pathway:A porcine model

目的基于转化生长因子β1(TGF-β_(1))/Smads/α-肌动蛋白-2(ACTA2)信号通路探讨猪食管内镜黏膜下剥离术(ESD)环切术后人工溃疡纤维化的机制。方法将8头实验白猪分为假手术组(n=4)与模型组(n=4),模型组通过ESD环切术建立猪食管狭窄模型。术后第3周分别采集两组猪食管组织,采用HE染色观察食管组织的病理特征,采用实时荧光定量PCR检测TGF-β_(1)、Smad2、Smad3、ACTA2 mRNA的相对表达水平,采用免疫组化法检测结缔组织生长因子(CTGF)、ACTA2的阳性表达情况,采用Western blotting检测TGF-β_(1)、Smad2/3、p-Smad2/3、Smad4、Smad7、CTGF、ACTA2蛋白的相对表达水平。结果与假手术组比较,模型组人工溃疡部位成纤维细胞(FB)大量增殖,并向肌成纤维细胞(MFB)表型转化。实时荧光定量PCR显示,与假手术组比较,模型组TGF-β_(1)(P<0.001)、Smad3(P=0.004)、ACTA2(P=0.001)mRNA表达均明显上调。免疫组化结果显示,与假手术组比较,模型组CTGF(P<0.001)、ACTA2(P<0.001)的表达明显升高。Western blotting结果显示,与假手术组比较,模型组TGF-β_(1)(P=0.002)、Smad2/3(P=0.003)、p-Smad2/3(P=0.002)、Smad4(P<0.001)、Smad7(P=0.016)、CTGF(P<0.001)、ACTA2(P<0.001)蛋白表达均明显上调,差异有统计学意义。结论成功建立ESD环切术后猪食管狭窄模型,该模型人工溃疡纤维化的形成可能与TGF-β_(1)/Smads/ACTA2信号通路相关。

Objective To investigate the mechanism of artificial ulcer fibrosis of stenosis after full circumcision of esophageal mucosal dissection in porcine model based on TGF-β_(1)/Smads/ACTA2 signaling pathway.Methods A total of eight pigs were randomized into two groups(4 in each group):sham operation group and model group.Animals in the model group received full circumcision of ESD to establish the esophageal artificial ulcer fibrosis model.Three weeks after the surgery,we collected esophageal tissues from animals.We further analyzed the tissues with Hematoxylin-eosin(HE)staining to observe the pathological characteristics of esophageal tissues.The real-time quantitative polymerase chain reaction(qRT-PCR)was employed to detect the mRNA relative expression levels of TGF-β_(1),Smad2,Smad3,and ACTA2.We then examined the positive expression of CTGF and ACTA2 using immunohistochemistry.Lastly,we detected the protein relative expression levels of TGF-β_(1),Smad2/3,p-Smad2/3,Smad4,Smad7,CTGF,and ACTA2 using Western blotting.Results Compared with the sham operation group,the fibroblasts in the artificial ulcer site proliferated rapidly with a more transformed myofibroblast phenotype in the model group.In the model group,the qRT-PCR results showed up-regulated mRNA levels of TGF-β_(1)(P<0.001),Smad3(P=0.004),and ACTA2(P=0.001).The results of immunohistochemistry showed that the positive expression of CTGF(P<0.001)and ACTA2(P<0.001)in the model group were higher than those in the sham operation group.We also observed up-regulated levels of TGF-β_(1)(P=0.002),Smad2/3(P=0.003),p-Smad2/3(P=0.002),Smad4(P<0.001),Smad7(P=0.016),CTGF(P<0.001),and ACTA2 in the model group,compared with the sham operation group.Conclusion A porcine model of stenosis after full circumcision of esophageal mucosal dissection was successfully established,and the mechanism may be related to the artificial ulcer fibrosis by TGF-β_(1)/Smads/ACTA2 signaling pathway.