肝富集转录因子在HBV基因转录与复制中的作用研究  

On the Role of Liver-enriched Transcription Factors in Regulating HBV Transcription and Replication

作　　者：王松[1] 唐红[2] 何芳[2] 刘丽[2] 黄飞骏[3] 周陶友[2] 赵连三[2] 

机构地区：[1]深圳市东湖医院,深圳518020 [2]四川大学华西医院感染性疾病中心生物治疗国家重点实验室 [3]四川大学华西基础医学与法医学院法医病理学教研室

出　　处：《四川大学学报（医学版）》2006年第1期35-39,共5页Journal of Sichuan University(Medical Sciences)

基　　金：国家杰出青年基金(资助号30325036)资助

摘　　要：目的观察各种肝富集转录因子对乙型肝炎病毒(HBV)基因转录和病毒复制水平的影响,探讨其在HBV嗜肝性机理中的作用。方法用复制型HBV重组质粒PHBV4.1与肝富集转录因子HNF1、HNF3、HNF4、HNF6、C/EBP或RXRΑ/PPARΑ的表达质粒,分别共转染非肝源细胞株NIH3T3、HELA、293T、SW1353、CV-1和COS1。用NORTHERN吸印杂交检测HBV3.5KB、2.4/2.1KB、0.7KB MRNA的转录情况,SOUTHERN吸印杂交检测HBV DNA复制中间体水平。结果用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3.5KB HBV RNA转录,也无HBV DNA复制;当用肝富集转录因子共转染时,HNF4和RXRΑ/PPARΑ的表达能够激活3.5KB HBV RNA转录和HBV DNA复制,而HNF1、HNF3、HNF6和C/EBP未能激活HBV复制。在HELA、293T、SW1353、CV-1和COS1细胞中,HNF4、RXRΑ/PPARΑ对HBV的转录和复制也具激活作用。进一步用突变型HBV重组质粒研究发现,C启动子HNF4结合位点的突变明显减弱了HNF4和RXRΑ/PPARΑ对HBV复制的激活作用。结论肝富集转录因子HNF4和RXRΑ/PPARΑ可支持HBV在非肝源细胞中的转录与复制;肝特异性基因转录是HBV嗜肝性的决定因素之一。Objective To investigate the effects of various liver-enriched transcription factors in regulating HBV transcription and replication, and to explore their potential roles in HBV hepatotropism. Methods The replication-competent HBV recombinant plasmid pHBV4.1 plus different liver-enriched transcription factor （HNF1 ,HNF3,HNF4,HNF6,C/EBP and RXRα/PPARα） expression plasmids were co-transfected into nonhepatic cell lines （NIH3T3,HeLa,293T,SW1353,CV-1 and COS1）. The transcription levels of 3. 5 kb,2.4/2. 1 kb and 0.7 kb HBV RNA were analyzed by Northern blot hybridization, and the level of HBV DNA replication intermediates was detected by Southern blot hybridization analysis. Results In the absence of co-transfected liver enriched transcription factor expression vectors, the 3. 5 kb HBV RNA is not transcribed and HBV DNA replication is not detected after transfecting of NIH 3T3 cells with pHBV4.1. Expression of the liver-enriched transcription factor HNF4 or RXRα/PPARα, stimulates the transcription of 3. 5 kb HBV RNA and the replication of HBV DNA. In contrast, expression of HNF1 ,HNF3,HNF6 and C/EBP does not stimulate the transcription of 3. 5 kb HBV RNA and therefore does not activate viral replication. HNF4 and RXRα/PPARα were also shown to activate the transcription of 3.5 kb HBV RNA and viral replication in divers cell types including HeLa,293T, SW1353,CV-1 and COS1 cells. Mutation of the proximal nucleocapsid HNF4 binding site results in a greatly decreased level of HNF4 or RXRα/PPARαdependent HBV replication. Conclusion This study demonstrated that the liver-enriched transcription factors HNF4 and RXRα/PPARα can support HBV transcription and replication in nonhepatic cells, indicating that liver-specific gene transcription is one of the determinants of HBV hepatotropism.

关 键 词：乙型肝炎病毒 肝富集转录因子 转录 复制 嗜肝性 

分 类 号：R373.2[医药卫生—病原生物学]