| Summary: | 博士 === 長庚大學 === 臨床醫學研究所 === 101 === Biliary atresia (BA) is the major cause of prolonged cholestasis in infancy characterized by idiopathic, complete obliteration of the extrahepatic bile duct. Kasai procedure is the only effective surgical intervention for correction of BA except for liver transplantation, but most patients still develop liver fibrosis and finally cirrhosis. The initiation of liver fibrosis is commonly shared by various liver disorders by activation of hepatic stellate cells (HSCs) and Kupffer cells, as well as synthesis and deposition of extracellular matrix components. Matrix metalloproteinases (MMPs) are the proteases responsible for tissue remodeling that play an important role in the progression of liver fibrosis.
To explore the major MMPs involving in BA-associated liver fibrosis, we analyzed MMP-2, -7, -9 and -13 mRNA expression in the liver tissue of early stage BA at the time of Kasai’s procedure (KP), late stage BA at the time of liver transplantation with advanced fibrosis (LT), and non-diseased control without liver fibrosis. The results showed that MMP-2 expression was significantly higher in LT group than both in control and KP groups, and MMP-7 expression was sequentially elevated when comparing control, KP and LT groups with significant difference between control and LT groups. Moreover, the fold difference between groups in MMP-7 mRNA was much higher than that in MMP-2. Immunohistochemical analysis revealed a significant positive correlation of MMP-7 expression with the stages of liver fibrosis. In situ hybridization demonstrated that the bile ductular epithelial cells, Kupffer cells and hepatocytes were the major producers of MMP-7 in liver. Our results imply that MMP-7 is a major MMP associated with the tissue remodeling during the progression of liver fibrosis in BA.
To analyze the cytokine expression during the progression of liver fibrosis in BA, a cDNA array was used followed by real-time quantitative RT-PCR for comparison between KP and LT. The result showed that Delta-like 1 homolog (DLK1), a novel gene known as a negative regulator of adipocyte differentiation, was up regulated in KP and down regulated in LT, similar to the expression pattern of Procollagen α1(I). Further characterization with immunohistochemistry, confocal microscopy, and in situ hybridization revealed that the DLK1 protein was mainly present in the cytoplasm of SMA-positive mesenchymal cells identical to activated HSCs, whereas the DLK1 mRNA was present only in hepatocytes. DLK1 has been proposed to act as a regulator during cell differentiation in a variety of tissues and most likely a suppressor of cell differentiation. Accordingly, we hypothesize that DLK1 overexpression in KP may implied its participation in the regulation of HSC transformation from quiescent fat-storing cells to myofibroblasts in BA-associated fibrogenesis.
To perform functional study of DLK1, we generated recombinant protein of DLK1 extracellular domain (DLK1-EC) to treat in-vitro activated HSCs and an HSC cell line, HSC-T6. The results revealed that cell migration but not proliferation were significantly inhibited in both activated HSC and HSC-T6 with down regulation of fibrogenic markers, including α-SMA and fibronectin, by DLK1-EC treatment. In conjunction with a recent report showing that DLK1-EC is able to promote activation of HSCs during chronic liver injury, the expression of DLK1 may be critical for the activation of HSCs but may conversely inhibit the perpetuation of HSCs in liver fibrosis.
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