Wang Robert Yung-Liang, Shen Chia-Ning, Lin Min-Hui, Tosh David, Shih Chiaho
Department of Pathology, University of Texas Medical Branch, Galveston, 77555-0609, USA.
J Virol. 2005 Oct;79(20):13116-28. doi: 10.1128/JVI.79.20.13116-13128.2005.
Recently, a rat pancreatic cell line (AR42J-B13) was shown to transdifferentiate to hepatocyte-like cells upon induction with dexamethasone (Dex). The aim of this study is to determine whether transdifferentiated hepatocytes can indeed function like bona fide liver cells and support replication of hepatotropic hepatitis B virus (HBV). We stably transfected AR42J-B13 cells with HBV DNA and examined the expression of hepatocyte markers and viral activities in control and transdifferentiated cells. A full spectrum of HBV replicative intermediates, including covalently closed circular DNA (cccDNA) and Dane particles, were detected only after induction with Dex and oncostatin M. Strikingly, the small envelope protein and RNA of HBV were increased by 40- to 100-fold upon induction. When HBV RNAs were examined by primer extension analysis, novel core- and precore-specific transcripts were induced by Dex which initiated at nucleotide (nt) 1820 and nt 1789, respectively. Most surprisingly, another species of core-specific RNA, which initiates at nt 1825, is always present at almost equal intensity before and after Dex treatment, a result consistent with Northern blot analysis. The fact that HBV core protein is dramatically produced only after transdifferentiation suggests the possibility of both transcriptional and translational regulation of HBV core antigen in HBV-transfected AR42J-B13 cells. Upon withdrawal of Dex, HBV replication and gene expression decreased rapidly-less than 50% of the cccDNA remained detectable in 1.5 days. Our studies demonstrate that the transdifferentiated AR42J-B13 cells can function like bona fide hepatocytes. This system offers a new opportunity for basic research of virus-host interactions and pancreatic transdifferentiation.
最近,一种大鼠胰腺细胞系(AR42J-B13)被证明在用地塞米松(Dex)诱导后可转分化为肝细胞样细胞。本研究的目的是确定转分化的肝细胞是否真的能像真正的肝细胞一样发挥功能,并支持嗜肝性乙型肝炎病毒(HBV)的复制。我们用HBV DNA稳定转染AR42J-B13细胞,并检测对照细胞和转分化细胞中肝细胞标志物的表达及病毒活性。仅在用地塞米松和制瘤素M诱导后,才检测到包括共价闭合环状DNA(cccDNA)和丹氏颗粒在内的全套HBV复制中间体。引人注目的是,诱导后HBV的小包膜蛋白和RNA增加了40至100倍。当通过引物延伸分析检测HBV RNA时,地塞米松诱导产生了分别起始于核苷酸(nt)1820和nt 1789的新型核心和前核心特异性转录本。最令人惊讶的是,另一种起始于nt 1825的核心特异性RNA在Dex处理前后的强度几乎始终相等,这一结果与Northern印迹分析一致。仅在转分化后才大量产生HBV核心蛋白这一事实表明,在HBV转染的AR42J-B13细胞中,HBV核心抗原可能存在转录和翻译调控。撤去地塞米松后,HBV复制和基因表达迅速下降——1.5天内可检测到的cccDNA不到50%。我们的研究表明,转分化的AR42J-B13细胞可以像真正的肝细胞一样发挥功能。该系统为病毒-宿主相互作用和胰腺转分化的基础研究提供了新的机会。