胆管对端吻合术后胆管纤维化的动物实验研究

Animal experimental study of biliary fibrosis in end to end anastomosis after transection of the common bile duct

目的探讨胆管对端吻合术后胆管纤维化的机制。方法将12头6~8个月龄、体重30~40 kg的内江小香猪分为实验组和对照组,每组6头。实验组胆总管横断伤后行对端吻合术,对照组为假手术组,仅行T管置入。术后9个月采用Masson染色、荧光定量聚合酶链反应、免疫组化对比分析吻合口胆管组织中促纤维化因子转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)和上皮间质转化(EMT)标志物细胞角蛋白-19(CK19)、E-钙粘蛋白(E-Cad)、纤维母细胞特异蛋白-1(S100A4)、α-平滑肌肌动蛋白(α-SMA)以及胶原成分胶原I(COL-1)、胶原III(COL-3)和纤连蛋白(FN)含量的表达变化。结果 Masson染色表明,实验组胆管黏膜下胶原纤维增多。与对照组相比,实验组吻合口胆管组织中主要纤维化指标TGF-β1[(3.482±0.313)比(1.000±0.102),t=18.43,P<0.001]和CTGF[(2.160±0.287)比(1.000±0.103),t=9.32,P<0.001]mRNA表达增加,差异具有统计学意义。实验组EMT标志物CK19和E-Cad的mRNA和蛋白表达均比对照组下降,而S100A4和α-SMA的mRNA和蛋白表达均比对照组增加,差异均具有统计学意义(均P<0.01)。结论胆总管横断伤后即时行对端吻合术可行,但长期观察吻合口胆管组织内存在纤维化。

Objective To explore the mechanism of biliary fibrosis after end to end anastomosis of bile duct.Methods 12 Neijiang miniature pigs(6-8 months old,weight 30-40 kg)were divided into experimental group(n=6)and control group(n=6).The model of end to end anastomosis after transection of the common bile duct was established in experimental group.Control group was a sham operation group,and only T tube was placed.After 9 months,Masson staining,fluorescence quantitative PCR and immunohistochemistry were used to analyze the expressing changes of pro-fibrotic factor transforming growth factor-β1(TGF-β1),connective tissue growth factor(CTGF)and epithelial-mesenchymal transition(EMT)markers,including cytokeratin-19(CK19),E-cadherin(E-Cad)and fibroblast specific protein-1(S100A4),α-smooth muscle actin(α-SMA)and collagen components Collagen I(COL-1),collagen III(COL-3)and fibronectin(FN)in the anastomotic bile duct tissues.Results Masson staining showed that the submucosal collagen fibers increased significantly in the experimental group.Compared with the control group,the mRNA expression of TGF-β1[(3.482±0.313)vs.(1.000±0.102),t=18.43,P<0.001],CTGF[(2.160±0.287)vs.(1.000±0.103),t=9.32,P<0.001]were increased,the difference was statistically significant.Compared with control group,the mRNA and protein expression of CK19 and E-Cad were decreased in the experimental group,while the mRNA and protein expression of S100A4 andα-SMA were increased in the experimental group(all P<0.01).Conclusion It was feasible in the short term to perform an end-to-end anastomosis after transection of the common bile duct,but there was an obvious fibrosis in the anastomotic bile duct tissue at later time.