肝內膽道癌為第二種常見的形式在肝臟癌症中僅次於肝細胞癌。我們先前的研究指出在斑馬魚肝臟專一性的表達 HBV X 蛋白和 HCV core蛋白會導致肝內膽管癌的產生，並且證實TGF-β1扮演重要角色，雖然TGF-β1下游的p38與Smad3L在肝內膽道癌中有活化，然而，其中的TGF-β1是否在參與誘發肝內膽管癌時是透過 JNK 或p38來調控Smad3L磷酸化的機制尚未清楚。我們過去的結果也顯示在一個月雙重表現HBx和HCP蛋白的斑馬魚肝臟中有初期的肝臟膽管病變。我們利用一個月的雙重轉基因斑馬魚從組織學和基因學上來分析ICC生成原因。利用免疫組織化學染色和次世代基因定序透過MetaCore軟體來分析，結果中我們可以發現GeneGo分析TGF-beta1影響細胞生長週期、發育和細胞骨架重建排在第一位。TGF-β1所影響的EMT和細胞增生也扮演重要的角色。QRT-PCR結果也顯示TGF-β1/Smad3L的活化.同時我們也利用PCNA抗體發現在一個月的雙重轉基因斑馬魚肝臟有高度的細胞增生現象。利用HuCCT1人類膽道癌細胞株,我們發現了TGF-β1會直接活化JNK與p38，進一步影響Smad3 連接位置的磷酸化。從以上結果顯示TGF-β1/Smad3L的活化對於ICC的初形成可能扮演了重要的角色。
Intrahepatic Cholangiocarcinoma (ICC) is the second most common type of primary liver cancer after hepatocellular carcinoma (HCC). Our previous results demonstrated that liver-specific co-expression of HBx and HCP liver induce ICC formation in zebrafish. In which, TGF-β1 play an important in cholangiocarcinogenesis. Although TGF-β1 downstream protein p38 and Smad3L were phosphorylated in ICC. However, whether TGF-β1 contributes to the initiation of ICC through pJNK or pp38 mediated phosphorylation of Smad3L is still unclear. Our previous results also found that the liver of one-month-old HBx+HCP transgenic zebrafish exhibit several predominant feature of hepatobiliary disorder. To investigate the initiation mechanism of ICC, we histologically and genetically analyzed the liver of one-month-old zebrafish by IHC and next generation sequencing (DGE), respectively. The results show that the different gene expression profiles in the liver include genes involved in cell cycle initiation, development and cytoskeletal remodeling among the top 10 GeneGo pathways at one-month-old zebrafish. In which, TGF-β-dependent epithelial-mesenchymal transition (EMT) and cell proliferation occurred among the top 10 rankings. QRT-PCR confirmed the activation of TGF-β/Smad3L pathway. Using an antibody against PCNA, we found that liver of HBx+HCP zebrafish with dilated bile ducts showing increased proliferation. To investigate the role of TGF-β1/Smad3L pathway in intrahepatic cholangiocarcinogenesis, we treated human ICC cell line HuCCT1 with TGF-β1. Results revealed that TGF-β1 induced Smad3 linker region phosphorylation through pJNK and pp38 in HuCCT1 cells. Taken together, TGF-β1/Smad3L pathway may play an important role in HBx- and HCP-induced ICC development.