摘要
为研究NIRF(Np95/ICBP-90 like RING finger protein)对乙型肝炎病毒(hepatitis B virus,HBV)的复制以及与乙型肝炎病毒共价环状闭合DNA(HBV cccDNA)结合的组蛋白H3乙酰化的影响,采用脂质体转染将pGEM-HBV1.3+pFLAG、pGEM-HBV1.3+pFLAG-NIRF、pGEM-HBV1.3质粒分别转入HepG2细胞,Western blot检测NIRF蛋白的表达,用ELISA结合RT-PCR检测HBsAg、HBeAg以及HBV cccDNA的量并同时说明HBV在细胞内是完成完整复制表达的,采用染色质免疫共沉淀(ChIP)的方法检测与HBV cccDNA结合的组蛋白H3以及H3乙酰化水平的动态变化。结果显示,NIRF蛋白下调HBV标志物HBeAg、HBsAg的分泌以及HBV cccDNA的表达,表明其对HBV复制具有抑制作用;组蛋白H3及乙酰化的组蛋白H3都与HBV cccDNA的动态变化水平呈现相似的平行性,而NIRF蛋白也明显抑制组蛋白H3的表达水平和乙酰化水平。结论证实NIRF不仅能抑制HBV在肝癌细胞中的复制,而且能下调与HBV cccDNA结合的组蛋白H3和乙酰化组蛋白H3的表达。期待NIRF能为后续的HBV致病机理、HBV复制表观遗传学水平研究及有效抗病毒药物的研究与开发提供理论上的支持与帮助。
To determine the effect of NIRF protein （Np95/ICBP-90 like RING finger protein） on the rep- lication of HBV and the acetylation of cccDNA-bound H3 histone at various times after transient transfection of linear HBV DNA into human hepatoma HepG2 cells. HepG2 cells were infected with GEM-HBV1.3＋pFLAG, pGEM-HBV1.3＋pFLAG-NIRF, pGEM-HBV1.3 plasmids. The secretion of HBsAg and HBeAg in the cultural supematants of the transfected cells was detected by ELISA. The expression of HBV mRNA and NIRF protein was examined by RT-PCR and Western blot, respectively. Finally the levels of the HBV cccDNA-bound H3 histone and the acetylated H3 histone were identified by ChIP （Chromatin Immunoprecipitation）. Our results demonstrated that the acetylation of cccDNA-bound H3 histone was associated with the level of HBV replication. NIRF can inhibit the levels of cccDNA-bound H3 and acetylated H3 in HepG2 cells, which may play roles in a better understanding of the mechanisms of HBV and confirm new therapeutic strategies against hepatitis B virus.
出处
《中国细胞生物学学报》
CAS
CSCD
北大核心
2013年第3期322-327,共6页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:30872248)
重庆市科技委员会基金(批准号:2008BB5400)
重庆市教育委员会基金(批准号:KJ080326)资助的课题~~
