Transgenic HBV X and HCV Core co-expression Leads to Liver Inflammation and Fibrosis in Zebrafish

在臨床研究上，雙重感染B型肝炎與C型肝炎將提高產生嚴重急性肝炎、肝硬化與肝癌的風險。而B型肝炎X蛋白與C型肝炎核心蛋白均已被證實與肝臟硬化相關，且為肝癌產生的重要因素。本研究目的旨在建立肝臟專一表現HBV x與HCV croe雙重轉殖斑馬魚，用以探討研究雙重感染下的致病機制以及腫瘤形成之分子機制。本研究中，我們利用四環黴素誘導表現系統，來調控轉殖基因的表現，此系統包含兩個載體，一為利用肝臟專一表現肝臟型脂肪酸結合蛋白啟動子驅動遠端四環黴素激活調控因子表現，另一載體則具有四環黴素誘導反應子配合雙向巨細胞病毒啟動子，可同時表現出轉殖基因與報導基因,如綠色螢光蛋白。另配合Tol2轉座因子系統可有效提昇基因轉殖的效率。在組織學的分析上，我們發現了約有30%的雙重轉殖斑馬魚在一個半月時具有病徵的出現（20%為發炎，10％為肝纖維化現象）。而在三個月成熟轉基因魚則發現纖維化現象比例增加約為30%，且於較嚴重的個體，發現具有惡性腫瘤的形成。但是，我們在控制組及單一轉殖HBV x與HCV core蛋白轉基因魚則無發現任何病理徵狀。進一步，我們運用定量PCR分析肝臟纖維化相關基因，結果顯示，在我們的模式動物上，發炎及引起細胞壞死之相關基因（TGF-β、TNFα、NFκB、Cox2a）表現均明顯的上昇，且造成TGF-β下游基因CTGF(結締組織生長因子)的過量表現，而且CTGF的表現量與肝纖維化的程度呈正相關。此外，四環黴素誘導處理雙重轉殖魚使得HCP與HBx表現下降也使得CTGF基因表現被向下調控，因此，CTGF過度表現是受到HCP與HBx的調控所致。此顯示出，CTGF在雙重轉殖所造成肝臟纖維化的分子機制上扮演著重要的角色。總結，這是第一個運用雙重轉殖肝炎病毒蛋白導致癌化的模式動物，此動物模式將可提供未來用於研究HBV x與HCV core交互作用之致病機轉。Epidemically, co-infection with hepatitis B and C can significantly increase the risk of development of fulminant hepatitis, cirrhosis and hepatocelluar carcinoma(HCC). HBV x (HBx) and HCV core proteins (HCP) are associated with liver cirrhosis and represent major risk factors for HCC development. We aim to establish liver-specific HBx and HCP double transgenic zebrafish to study the dual infection pathogenesis and molecular mechanism in tumor formation. Here, we use tetracycline inducible expression system (tetracycline-off system) which can conditionally express transgenes, involving two vectors: one contains tTA(trans-tetracycline activator) driven by liver specific L-FABP (liver-type fatty acid binding protein) promoter, another with TRE(tetracycline responding element) site flanked by bidirectional CMV promoter to simultaneously express transgenes and reporter genes, such as GFP. Combining with Tol2 transposon system can increase efficiency of transgensis. Histological evidences shows about 30% pathological occurrence (20% inflammation and 10% fibrosis) in 1.5-month-old double transgenic fish, and the fibrosis ratio has been increased in 3–month-old adult fish about 30%. Even found malignancy tumour formation in severe fibrosis fish. However, compared to the control、no significant pathological change was observed in single transgenic zebrafish. Further, we performed quantitative RT-PCR to analysis fibrosis-related genes. Results showed that several inflammation and cell necrosis marker genes (TGF-β、TNFα、NFκB、Cox2a) were highly expressed, and the expression level of CTGF, a mediator of the profibrotic activities of TGFβ1 (a known regulator of fibrosis) is correlated with the degree of fibrosis phenotype, regulate by HCP and HBx. These findings might suggest that connective tissue growth factor (CTGF) play an important role in co-expression inducing liver fibrosis mechanism. In conclusion, this is the first liver tumorigenesis animal model with hepatitis B and C viral proteins co-expression. Perspectively, this model can provide a good material for investigating the cross-talk between HCP and HBx.TABLE OF CONTENTShinese Abstract & keywords…………………………………………Inglish Abstract & keywords………………………………………... IIntroduction……………………………………………………………..1aterial and Methods…………………………………………………..9esults…………………………………………………………………..17Establishment of inducible HCV core protein and HBV x protein transgenic zebrafishEnhancement of transgenic line constructionIncrease transgenesis efficiency from introduction of tTA lineControl of doxcycline treatmentGeneration of HCP and HBX co-expression transgenic zebrafishCo-expresson of viral protein transgenic leads to hepatic severe inflammatory phenotype in 1.5mo-old zebrafishFibrosis phenotype occurred in co-expression transgenic zebrafish but not in single viral protein transgeneTGF-β signaling pathway was activated in fibrogenesis appearing of double transgenic zebrafishThe important role in pathological induction is based on whether the HCP and HBx expressed simultaneously in liverCTGF is an important regulator of fibrogenesis in HBx and HCP co-expression zebrafishTurn down HBX and HCP gene expression can contribute to CTGF down-regulation iscussion………………………………………………………………26eference……………………………………………………………….35igures………………………………………………………………….42ables…………………………………………………………………...7