| Objective: To observe the presence of angiogenesis and study the expression of angiogenesis related factors including vascular endothelial growth factor(VEGF),hypoxia-inducible factor-1α(HIF-1α),platelet-derived growth factor AA(PDGFAA),platelet-derived growth factor BB(PDGFBB),platelet-derived growth factor receptor α(PDGFRα)and platelet-derived growth factor receptor β(PDGFRβ)in the liver tissue of biliary atresia(BA)children,and to explore the role of each factor in the changes of BA angiogenesis and to analyze their relationship with the process of hepatic fibrosis.Methods: 1.Twenty patients with BA in Tianjin Children’s Hospital were selected as the Kasai group,another twenty patients including eight cholestasis and twelve biliary dilatation patients were selected as control group.Twenty BA patients with liver transplantation done in Tianjin First Central Hospital were selected as transplantation group.2.HE staining was used to observe the basic pathological changes of liver histology in three groups.According to Weidner’s methods to count the microvessel density(MVD)in the liver after CD34 staining marked blood vessels.According to Okamoto’s criteria to determine the degree of liver fibrosis after Masson staining marked the fibrous stroma.3.The protein and gene expression of VEGF,HIF-1α,PDGFAA,PDGFBB,PDGFRα and PDGFRβ in liver tissue of three groups were separately detected by immunohistochemical(IHC)staining and real-time fluorescence quantitative polymerase chain reaction(PCR).4.To compare the difference of vascular changes and the expression of angiogenesis related factors in the liver tissue of the three groups.To analysis the role of angiogenesis related factors in vascular changes and hepatic fibrosis process in BA.Results: 1.The presence of angiogenesis in three groups In control group,the lobular structure of the liver was close to normal,and the portal area was slightly wider.There was little infiltration of inflammatory cells,no fibrosis or mild fibrous tissue proliferation.In Kasai group,liver cells cholestasis,portal area widened,bile duct hyperplasia,the concentration of bile was seen,showing varying degrees of inflammatory cell infiltration and fibrous tissue hyperplasia.In transplantation group,liver cells necrosis,large number of abnormal hyperplasia of bile duct was seen,fake lobular formation.In Kasai group,the expression of CD34 was significantly increased in the portal area and the fibrous septa and also in the liver sinusoids that are lined by fenestrated and discontinuous endothelial cell.In transplantation group,CD34 was observed diffuse expression in the fibrous septa.The MVD was 16.8 ± 1.9,23.2±3.8 and 25.9±2.1 in the control,Kasai,and transplantation group,respectively(P<0.05).Compared with the control group,the MVD in Kasai and transplantation group were obvious increased(P<0.017).There was no significant difference between Kasai and transplantation group(P<0.017)..2.The expression of angiogenesis related factors in three groups Immunohistochemical results showed that the expression of VEGF in hepatocytes and cholangiocytes in the control group was moderate positive.The strong positive expression of VEGF was observed in these cells in the Kasai group and the transplantation group.In control group,HIF-1α was negative and was not expressed in most of the hepatocytes and only observed in a little liver cells.The expression of HIF-1α was strong positive in the cytoplasm and nuclear of hepatocytes in Kasai group and the transplantation group.In the control group,PDGFAA,PDGFBB,PDGFRα and PDGFRβ were weakly expressed only in some cells in the portal area,such as fibroblasts and macrophages,and was not expressed in bile duct cells and hepatocytes.PDGFRα were increased in the portal area in Kasai group,and was also expressed in the cholangiocytes.Both factors were decreased in the transplantation group.PDGFBB and PDGFRβ were increased in both portal area and cholangiocytes in Kasai group and the transplantation group.The results of IHC staining showed that the expression of VEGF,HIF-1α,PDGFBB,PDGFRα and PDGFRβ were significantly different between the three groups and the expression of Kasai and transplantation group was higher than that of the control group(P<0.05),except the PDGFAA.The expression of VEGF and PDGFBB in the transplantation group was higher than in the Kasai group,and the expression of PDGFRα was higher in the Kasai group than in the transplantation group(P<0.05).The results of PCR showed that all angiogenesis related factors were different in the three groups,and the expression of Kasai and transplantation group was higher than that of the control group(P<0.05).The expression of VEGF,HIF-1α and PDGFBB in the transplantation group was higher than that in the Kasai group,and the expression of PDGFRα was higher in the Kasai group than in the transplantation group(P<0.017).3.Correlation analysis of the protein expression of angiogenesis associated factor with angiogenesis and fibrosis There was a positive correlation between VEGF protein expression and microvessel density(P<0.05).HIF-1α,PDGFAA,PDGFBB,PDGFRα,PDGFRβ protein expression and microvessel density were not correlated.In addition,HIF-1α and VEGF protein expression was positively correlated(P<0.05).The expression of HIF-1α and PDGFBB was positively correlated with the degree of fibrosis of liver tissue in BA children(P<0.05).There was no correlation between VEGF,PDGFAA,PDGFRα and PDGFRβ and fibrosis.There was a positive correlation between microvessel density and fibrosis(P<0.05).Conclusions: There was angiogenesis in the liver of BA children,it is speculated that the up-regulation by hypoxia-induced HIF-1α-VEGF signaling pathway may be the mechanism of regulation of vascular proliferation in BA.PDGF family members play a different role in the different stages of BA fibrosia.The degree of vascular proliferation and fibrosis process consistency and interaction promote disease development in BA. |
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