| Biliary atresia is a common cause of the cholestatic jaundice in infancy, serious crisis babies and infants life. The disease incidence rate is from 1/14000-1/10000. On clinical, it is difficult to recognite with neonatal hepatitis. Although the Kasai surgical operation is popular, but 70%-80% of the operated children also had the progressive destruction of the bile duct and the liver fibrosis in following, finally needed liver transplantation. The etiology of this disease is unclear, more and more people consider that infection in perinatal is important in occurrence of BA, but it is also unclear that how dose the infection can happen progressive bile duct inflammation degeneration and liver fibrosis.Recently many researchers thinked that the epithelium-mesenchymal transition(EMT) as an important role in chronic fibrosis of tissues and organs. EMT refers to the polarization of epithelial cell differentiation and maturation of the loss of epithelial/endothelial cell characteristics, to the transformation of mesenchymal cells to greater activity and mobility, this process in embryonic development, tissue regeneration, tissues and organs fibrosis, the transfer of cancer cells play an important role. Physiological conditions, EMT is an important tissues and organs of the wound repair mechanism, tissues and organs after injury through the formation of fibrous scar EMT mechanisms to achieve wound healing, but this process was the persistence of pathological state of tissues and organs can cause fibrosis, sclerosis, this transformation mechanism in the capillary bronchi, renal tubules and other tubular fibrosis in the earlier study, reporting more. Recently EMT has been as an important therapeutic target to terminat, reversal of renal injury in progressive renal fibrosis. Now EMT was proposed as a mechanism behind the sclerosing cholangitis in BA, and in this way the bile duct epithelial cells gradually lose their characteristic, demonstrated characteristics of mesenchymal cells. Also this process involves a series of signal transduction pathways, including CK-19,Fibronectin,TGF-β,Smad3,Hes-1 and so on. Studies have shown that, CK-19 as a bile duct cell-specific cytokines,can directly response the pattern and the number changes of bile duct epithelial cells; Fibronectin as a mesenchymal cell-specific cytokines, can directly affect the formation of cytoskeletal components and assembly, enhance the adhesion capacity between cell-cell and cell and matrix and as an early signal in EMT; Transforming growth factor TGF-β1 is the strongest and the most cytokines in fibrosis, through the TGF-βsmad signaling to make the fibrosis process continu and further deterioration of fibrosis; Hes-1 is an important downstream signal in Notch signaling pathway, Notch pathway in the process of biological evolution of highly conservative, and involved in embryonic development, blood cell development and differentiation, T cells in thymic selection and the formation of blood vessels and other important physiological processes, and congenital abnormalities are closely related, Hes-1 expression by the Notch signal regulation However, the above indicators whether worked in children with biliary atresia and liver fibrosis in bile duct,and how to play a role, whether there is a link between them and other issues remain unclear.In this research, the study object are liver specimens respectively from biliary atresia children confirmed by surgery and no autopsy in children with congenital malformations. With the immunohistochemistry to observe the expression and distribution of the CK-19,Fibronectin,TGF-β1,Smad3 and Hes-1 in liver, discussion the role of epithelium-mesenchymal transition in biliary atresis, it is important for in-depth understanding the evolution of pathology in liver of BA.Method1.Specimen preparation and groupingThe preoperative ultrasound, MRCP, intraoperative findings, intraoperative cholangiography confirmed 25 cases of patients with biliary atresia as the case group, intraoperative sterile biopsy liver tissue is about 0.5cm×0.5cm×0.5cm size of the pathological specimens, with 4% paraformaldehyde fixation; the control group was no deformity in children the same age autopsy in 5 cases, liver tissue obtained, and with 4% paraformaldehyde. 2.ImmunohistochemistryImmunohistochemical methods were used to detect CK-19, Fibronectin, TGF-β, Smad3, and Hes-1 in the liver tissue of the control group and case group distribution and expression.3.Data AnalysisSPSS 11.0 software used for statistical analysis. Measurement data use by x±s.T test analysis was used to compare values. P<0.05 showing the differences were statistically significant.Result1. Bile duct epithelial cell-specific cytokines CK-19 in the BA group of liver expression of immunohistochemical staining significantly, a large number of capillary bile duct hyperplasia, bile duct lumen loss of normal structure, no fixed form, part of the phenotype of the loss of bile duct epithelial cells, form complex the distribution of disorder, and there is cholestasis significant structural changes in the lumen of the bile duct, bile duct epithelial cells in severe morphological changes. CK-19 in liver tissue of the case group immunohistochemical staining, the average number of bile duct epithelial positive cells (96.33±3.28/HP), more than the control group (20.58±1.15/ HP), the mean optical density (0.413±0.058), higher than the control group (0.264±0.021), compared with control group, P<0.001, the differences were statistically significant.2. An early indicator of fibrosis in the BA group of liver fibronectin immunohistochemical staining, the liver cells, the periportal fibrous tissue and bile duct epithelial cells were expressed, but in bile duct epithelial cells express significant. Fibronectin in liver tissue of the case group immunohistochemical staining, the average number of bile duct epithelial positive cells (57.24±2.08/HP), more than the control group (0/HP), P<0.001, the mean optical density (0.305±0.078), higher than the control group (0.093±0.001), P<0.001, the differences were statistically significant.3. Transforming growth factor TGF-β1 in children with BA immunohistochemical staining of liver tissue stained in parts of widely distributed in the liver cells, bile duct epithelial cells, portal area part of the fiber structure and its surrounding other cells, and the organizational structure of the surrounding liver cells staining more apparent and are mainly expressed in the cytoplasm than the bile duct epithelial cells express strong. TGF-β1 in liver tissue of the case group immunohistochemical staining, the average number of bile duct epithelial positive cells (85.16±2.65/HP), more than the control group (20.25±1.78/HP), the mean optical density (0.396±0.030), higher than the control group(0.241±0.021), compared with control group, P<0.001, the differences were statistically significant.4. Smad3 in the BA liver tissue in children with immunohistochemical staining in the liver cells and biliary epithelial cells exist in, and the bile duct epithelial cell staining was stronger than that of liver cells, stained area in the cell cytoplasm of periportal vascular smooth muscle cells also showed slight staining, individual bile duct lumen disappeared, bile duct epithelial cells lose their epithelial cell phenotype, transformed into mesenchymal cell phenotype, between integration, no separate structure. Smad3 in liver tissue of the case group immunohistochemical staining, the average number of bile duct epithelial positive cells (69.33±2.08/HP), more than the control group (11.53±2.15/HP), the mean optical density (0.398±0.035), higher than the control group(0.254±0.028), compared with control group, P<0.001, the differences were statistically significant.5. Hes-1 in liver tissues of BA in children with immunohistochemical staining, the bile duct epithelial cells, staining was stronger than the control group, staining mainly located in the cytoplasm, more epithelial cells showed mesenchymal cell phenotype, between adjacent cells, mutual integration, distribution disorder. Hes-1 in liver tissue of the case group immunohistochemical staining, the average number of bile duct epithelial positive cells (81.58±2.38/HP), more than the control group (12.97±2.73/HP), the mean optical density (0.408±0.063), higher than the control group(0.231±0.015), compared with control group, P<0.001, the differences were statistically significant.Conclusion1. BA liver tissue in patients with early stage a large number of bile canaliculi and bile duct epithelial cell proliferation, structure, morphology and distribution of disorder.2. Bile duct epithelial cells expressed mesenchymal cell-specific markers fibronectin, shows bile duct epithelial cells of the epithelial-mesenchymal transformation, EMT during biliary fibrosis play an important role.3. In the bile duct epithelial cells associated with EMT during embryonic development-related genes downstream of Notch signaling pathway re-activate, and accompanied by inflammation-related cytokines TGF-β1, Smad3 activation such as expression. |
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