【摘要】 目的建立乙型肝炎病毒(HBV)在非肝源细胞的复制体系,探讨肝富集转录因子对HBV基因转录和复制的调控作用。方法用复制型HBV重组质粒pHBV4.1与肝富集转录因子HNF3、HNF4和RXRα/PPARα的表达质粒分别共转染非肝源细胞株NIH3T3。用Northern吸印杂交检测HBV 3.5 kb、2.4/2.1 kb、0.7 kb mRNA的转录情况,Southern吸印杂交检测HBV DNA复制中间体水平。结果复制型HBV重组质粒pHBV4.1在肝癌细胞中能检测到3.5 kb HBV RNA转录产物和HBV DNA复制中间体;用pHBV4.1转染NIH3T3细胞后,在没有肝富集转录因子表达质粒共转染时未能检出3.5 kb HBV RNA转录产物,也无HBV DNA复制中间体;当用肝富集转录因子共转染时,HNF4和RXRα/PPARα的表达能够激活3.5 kb HBV RNA转录和HBV DNA复制,而HNF3未能激活HBV复制,但在HNF4或RXRα/PPARα介导的病毒复制体系中,均见3.5 kb HBV mRNA的转录和HBV DNA复制水平下降。结论利用肝富集转录因子成功地建立HBV在非肝源细胞中的转录与复制。其中,HNF4和RXRα/PPARα可支持HBV在非肝源细胞中转录与复制;HNF3抑制HBV肝特异性基因转录与复制。更多还原

【Abstract】 Objective To establish hepatitis B virus(HBV) replication system with nonhepatic cell lines and to study the regulatory role of various liver-enriched transcription factors in HBV gene transcription and replication.Methods The replication-competent HBV recombinant plasmid pHBV4.1 plus different liver-enriched transcription factor(HNF3,HNF4 and RXRα/PPARα) expression plasmids were co-transfected into nonhepatic cell lines respectively(NIH3T3).The transcription levels of 3.5 kb,2.4/2.1 kb and 0.7 kb HBV mRNA were analyzed by Northern blot hybridization,and the levels of HBV DNA replication intermediates were detected by Southern blot hybridization.Results 3.5 kb HBV RNA replication products and HBV DNA intermediates could be detected in hepatic cell transfected by replication-competent HBV recombinant plasmid pHBV4.1.However,in the absence of co-transfeeted liver-enriched transcription factor co-transfection,the 3.5 kb HBV RNA transcription products and HBV DNA replication intermediates could not be detected after transfection pHBV4.1 to NIH3T3 cells.Expression of HNF4 or RXRα/PPARα stimulated transcription of 3.5 kb HBV RNA and replication of HBV DNA with liver-enriched transcription factor co-transfection.However,HNF3 could not stimulate HBV replication.Transcription of 3.5 kb HBV RNA and replication of HBV DNA decreased in virus replication system transducted by HNF4 or RXRα/PPARα.Conclusion The HBV replication system with nonhepatic cell lines can be established with liver-enriched transcription factors.HNF4 and RXRα/PPARα support HBV transcription and replication in nonhepatic cells,and HNF3 inhibits specific hepatic gene transcription and replication.更多还原