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HCV NS3/4A基因外来表达对Huh7细胞凋亡及DNA损伤应答的影响 被引量:8

Over-expression of HCV NS3/4A Influences Cell Apoptosis and Cellular Response to DNA Damage
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摘要 NS3/4A是丙型肝炎病毒(hepatitis C virus,HCV)编码的丝氨酸蛋白酶复合体,是病毒完成自身复制周期的必要成分。该研究为调查NS3/4A对细胞凋亡及DNA损伤应答(DNA-damage response,DDR)的影响,在Huh7细胞中表达了外来NS3/4A基因。通过DAPI染色和MTT分析显示,外来表达NS3/4A显著诱导细胞的凋亡和增殖活力的下降。免疫荧光检测结果表明,NS3/4A可明显增加细胞内源性DNA双链断裂(double strand breaks,DSBs)损伤(γH2AX灶点升高);而进一步用X-ray诱导细胞外源性DSBs损伤后,外来表达NS3/4A的细胞显示出明显的DSBs损伤修复缺陷(减缓的γH2AX灶点消退)。免疫印迹法检测结果显示,NS3/4A可抑制喜树碱(Camptothecin,CPT)诱导的ATM第1 981位丝氨酸的磷酸化(pATM1 981)。以上结果提示,NS3/4A基因外来表达可引起细胞DNA损伤,抑制ATM介导的DSBs损伤修复信号,诱导细胞凋亡通路的活化。 NS3/4A is a proteases complex encoded by hepatitis C virus (HCV), which is essential for the replication cycle of HCV. In this study, to investigate the role of NS3/4A in cellular apoptosis and response to DNA damage, the HCV NS3/4A gene was over-expressed in Huh7 cells. The cellular apoptosis and viability were deter- mined by DAPI stains and MTT assay, and the results indicated that over-expression of NS3/4A could remarkably induce the apoptosis and decreased viability of Huh7 cells in vitro. The expression of yH2AX was determined by immunofluorescence (IF), and the result showed that NS3/4A could induce DNA double-strand breaks (DSBs) in Huh7 cells. In addition, the results also showed that over-expression of NS3/4A resulted in a marked DSB repair defect after treating cells with X-ray (prolonged existence of the yH2AX loci). Furthermore, we could also detect decreased activatory phosphorylation event (Serine 1 981) of the protein kinase Ataxia-telangiectasia mutated (ATM) after Camptothecin (CPT) treatment in NS3/4A expressed Huh7 cells by Western blot. Together, these results indi- cate that over-expression of NS3/4A gene can lead to increased levels of DNA damage, inhibition of DSBs repair mediated by ATM, and activation of the apoptosis pathway in Hnh7 cells.
出处 《中国细胞生物学学报》 CAS CSCD 北大核心 2014年第8期1110-1115,共6页 Chinese Journal of Cell Biology
基金 山西省自然科学基金(批准号:2014021037-9) 山西医科大学汾阳学院博士启动基金(批准号:1301)资助的课题~~
关键词 DNA损伤 丙型肝炎病毒 DNA双链断裂 非结构蛋白3 4A DNA damage HCV DNA double-strand breaks NS3.4A
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参考文献20

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