低密度cDNA Macroarray对干扰素α抗病毒蛋白的筛选

目的 基于低密度cDNA Macoarray技术筛选出差异表达的干扰素(IFN)α抗病毒基因,以探讨IFN α抗病毒蛋白的表达与HBV复制的关系. 方法 以一定浓度的IFN α处理肝胚瘤细胞株HepG2和HepG2.2.15细胞6h,用cDNA Macroarray分析比较两细胞株IFN α抗病毒基因表达谱,并筛选出差异表达的IFNα抗病毒基因.将表达HBV核心蛋白(HBc)的质粒pHBc-EGFP转染HepG2细胞,RT-PCR法分析HBc对IFN α抗病毒基因表达的影响.将表达抗黏病毒A蛋白(MxA)的表达质粒pcDNA3.1-Flag-MxA转染HepG2.2.15,以酶联免疫吸附试验、Dot blot、Southern blot等方法分别检测HepG2.2.15细胞表达释放的HBsAg与HBeAg、细胞外HBV DNA和细胞内HBV DNA复制中间体(松弛环状DNA、双股线性DNA),以判断HBV复制情况.两组间数据比较采用t检验,组间不同时间点数据比较采用单因素方差分析.结果 cDNA Macroarray分析显示HepG2和HepG2.2.15细胞的抗病毒基因表达谱具有差异性:IFNa抗病毒基因中干扰素诱导跨膜蛋白(IFITM)1、IFITM2、IFITM3、RING4等在HepG2.2.15细胞的表达被部分抑制,而重要的抗病毒蛋白MxA表达被完全抑制.HBc转染组细胞中MxA mRNA表达的相对水平为0.31±0.05,低于空白对照组的0.74±0.04,差异有统计学意义,P＜0.05.MxA蛋白转染HepG2.2.15细胞48、72 h后,MxA转染组细胞上清液中HBsAg的S/CO值分别为1.42+0.21和1.58±0.18,HBeAg的S/CO值为1.44±0.14和2.28±0.24,而空白对照组细胞上清液中HBsAg的S/CO值为1.92±0.19和2.79±0.25,HBeAg的S/CO值为2.31±0.46和3.37±0.29,两组细胞上清液中HBV抗原的S/CO值差异均有统计学意义,P值均＜0.05.细胞外HBV DNA、胞内HBV复制中间体DNA均无明显变化.结论 HBV及其抗原成分的复制和表达影响着IFNα抗病毒蛋白的表达;HBV通过抑制IFN α抗病毒蛋白的表达而发挥拮抗IFNα的抗病毒活性.

Based on the low-density cDNA Macroarray for screening of antiviral proteins of IFNa tissues

Objective To screen the gene expression profiles of IFN-α antiviral proteins based on a low-density cDNA Macroarray,and to explore the relationship between the expression of antiviral protein and the HBV replication.Methods The HepG2 and HepG2.2.15 cells were treated with various concentrations of IFN-α (0 IU/ml,100 IU/ml,1 000 IU/ml) of IFN-α for 6 h,and then the low-density cDNA Macroarray was used for analysing the expression profiles of antiviral genes and screening differential expressions of antiviral proteins.Meanwhile,the HepG2 cells were transiently transfected with HBV core protein-expressed plasmid pHBc-EGFP,and the expressions of antiviral proteins were analysed by RT-PCR assay.Moreover,the HepG2.2.15 cells were also transfected with the antiviral protein-expressed plasmid pcDNA3,l-Flag-MxA.ELISA was used for analysing the secreted HBV antigens,while dot blot and Southern blot were applied for analysing the extracellular HBV DNA and intracellular replicative intermediate HBV DNA in HepG2.2.15 cells.All data were presented as mean ± SD and analyzed using the t-test and one-way analysis of variance (ANOVA)in the experiments.Results The Macroarray results suggested that the expression of IFN-α antiviral genes like 6-16,IFITM1,IFITM2,IFITM3 and RING4 in HepG2.2.15 cells were partially inhibited.More importantly,it was found,in this research,the expression of antiviral protein MxA in HepG2.2.15 cells was completely suppressed.RT-PCR analysis indicated that the expression of MxA was also significantly decreased in HepG2cells transfected with pHBc-EGFP plasmid.Although HepG2.2.15 cells transfected with pcDNA3.1-Flag-MxA plasmid could not inhibit extracellular HBV DNA and intracellular replicative intermediate HBV DNA,the MxA exerted some antiviral activities as it effectively suppressed the secretion of HBsAg and HBeAg in HepG2.2.15cells.Conclusions HBV and its antigen components probably influence the expression of antiviral proteins.IFN- resistance may be related to the down-regulation of antiviral proteins expression.