P-Smad 3在胆道闭锁肝纤维化中的作用机制研究

目的研究转化生长因子-β1(Transforming growth factor-β1,TGF-β1)、促纤维化通路中TGF-β1、P-Smad3等通路蛋白在胆道闭锁(Biliary atresia,BA)肝纤维化进程中的作用机制。方法选取2010年1月到2015年7月我们收治的胆管扩张症肝活检病例10例,为胆扩组;BA肝活检病例19例,为Kasai组;BA晚期肝移植患者自体肝活检病例11例,为移植组11例。进行苏木素/伊红(HE)、马松(Masson)染色观察肝标本纤维化评价其纤维化程度,免疫组化(Immunohistochemistry,IHC)染色检测TGF-β1、Smad2-4、P-Smad2-3及人纤溶酶原激活物抑制因子1(PAI-1)在肝组织中的表达,结合BA肝纤维化病理分级标准探讨TGF-β1等通路蛋白在肝纤维化进程中的作用。结果 HE及Masson:胆扩组偶见纤维细胞增生,Kasai组胶原纤维增生、桥接纤维化现象显著,移植组假小叶显著。免疫组化:①定性分析:胆扩组肝内Smad 2、Smad 4、PAI-1为阳性表达,其余为弱阳性;Kasai组TGF-β1等蛋白均在肝细胞胞质阳性表达,P-Smad 2、P-Smad 3、Smad4在胞核亦呈阳性表达;移植组患儿肝内TGF-β1等蛋白皆为弱阳性表达。②半定量分析:三组中Kasai组TGF-β1、Smad 3、P-Smad 3及PAI-1含量表达明显高于其他两组(P0.05),随着TGF-β1、Smad3、P-Smad3及PAI-1逐渐升高肝纤维化逐渐加重;PSmad3与PAI-1含量大小及变化幅度密切相关,促肝纤维化作用显著;Smad2、P-Smad2、Smad4组间无差异,在通路中连接相关蛋白。移植组TGF-β1等通路蛋白含量均少于Kasai组,随着肝硬化加重而逐渐减少。结论 BA肝内TGF-β1促纤维化通路中TGF-β1、Smad3显著促进肝纤维化,P-Smad3与肝纤维化进程密切相关。

The study on mechanism of p-smad3 in hepatic fibrosis of biliary atresia

Objetive To observe the function of TGF—β1 and P—Smad3 etc in TGF—β1 signal pathways in liver fibrosis of biliary atresia.Methods Liver biopsy specimens were collected from congenital biliary dila-tation (CBD group,n =10),biliary atresia patients who had Kasai procedure (Early hepatic fibrosis group,n =19),liver transplantation(transplantation group,n =11),the first two groups were collected from Jan.2010 to Jul.2015 in Tianjin childrens′hospital,the last group was collected from Jan.2013 to Jan.2014 in Tianjin first central hospital.The hematoxylin &eosin staining were used to observe the degree of liver fibrosis of every sin-gle sample,immunohistochemistry were used to observe the expression of TGF — β1、Smad 2 —4、P — Smad 2 ~3 and PAI—1 in liver tissues of these samples.Results HE and Masson:the CBD group had mild fiber cells hy-perplasia,the Kasai group had Proliferation of collagen fibers and bridging fibrosis phenomenon,the transplanta-tion group had significant pseudolobule.Immunohistochemistry:①qualitative analysis:in the CBD group,Smad 2,Smad 4,PAI — 1 expression is positive,the rest is weakly positive;in the Kasai group,the all proteins in the TGF —β1 signal pathways expressed in the liver cytoplasm,P— Smad 2,P — Smad 3,Smad 4 expression was also found in the nucleus;in the liver transplantation group,the expression of all proteins in the TGF — β1 signal pathways were weakly positive.②semi-quantitative analysis;The level of TGF—β1、Smad 3、P—Smad 3 and PAI— 1 in the Kasai group have a significant rise than the other groups(P <0.05),the hepatic fibrosis is becoming serious with the rise of TGF —β1、Smad 3、P—Smad 3 and PAI — 1,at the same time The correlation between P-Smad 3 and PAI — 1 was remarkable.there were no difference of Smad 2、P — Smad 2、Smad 4 between these groups(P >0.05).All of the proteins in the TGF —β1 signal pathways were decreased in transplantation group (P <0.05). Conclusions TGF—β1 and Smad 3 made a contribution to liver fibrosis in the early stage of bili-ary atresia,and P—Smad 3 is closely related with the progress of liver fibrosis.