摘要
干扰素α(IFN-α)是临床最常用的抗乙型肝炎病毒(HBV)药物之一。泛素特异性蛋白酶18(USP18)被证实是抑制IFN-α抗HBV活性的因子,但USP18是否对干扰素λ(IFN-λ)抗HBV有影响还尚未可知。为了明确USP18对IFNλ抗HBV活性的影响,本研究以Hep G2. 2. 15细胞作为乙肝体外模型,采用脂质体转染法分别向细胞转染p EGFP-USP18、PEGFP-N1经48 h,再经IFN-α和IFN-λ处理24 h,分为阴性对照组﹑USP18过表达+IFN-α组﹑空载组+IFN-α组﹑USP18过表达+IFN-λ组﹑空载组+IFN-λ组。采用Western印迹、RT-q PCR和ELISA检测各组的乙肝病毒标志物、STAT1/p STAT1和下游的干扰素刺激基因(ISGs)的表达。结果显示,与阴性对照组和空载组相比,USP18蛋白在过表达组明显升高(P <0. 05),过表达细胞模型构建成功;在IFN-α处理的两组中,空载组中HBs Ag、HBe Ag、HBc Ag及HBV-DNA的表达均低于USP18过表达组,差异有统计学意义(P <0. 05)。而IFN-λ处理组中,乙肝病毒标志物的差异不明显。在IFN-α处理组中,空载组的ISG15、Mx A、IFIT1和p STAT1表达均高于USP18过表达组,差异有统计学意义(P <0. 05),而在IFN-λ处理组中ISGs和p STAT1的表达无明显差异。上述结果证实,USP18可通过抑制JAK/STAT信号通路的激活来减弱IFN-α抗HBV的活性。研究还证实,IFN-λ可发挥抗HBV的作用,USP18不通过JAK/STAT信号通路抑制其抗HBV活性。
Interferon alpha( IFN-α) is one of the most widely used anti-hepatitis B virus( HBV)medicine. It has been reported that ubiquitin specific protease 18( USP18) inhibits the anti-HBV activity of IFN-α,but whether USP18 has an effect on the anti-HBV activity of IFN-λ was unclear. Here we aim to detect the anti-HBV effect of USP18 on IFN-λ in an in vitro model. HepG2. 2. 15 cells were transfected with two different plasmids: the empty vector( p EGFP-N1) and USP18 overexpressed plasmid( p EGFPUSP18),and then were treated with IFN-α and IFN-λ for 24 hours,respectively. Untreated groups served as the negative control. The expression of HBV markers,STAT1/p STAT1 protein expression and interferon stimulated genes( ISGs) were tested on HepG2. 2. 15 by Western blotting,quantitative realtime PCR( RT-q PCR) and enzyme-linked immunosorbent assays( ELISA). The results showed that USP18 was successfully overexpressed in the overexpression group compared with both negative control and empty vector control groups( P < 0. 05),which demonstrated the successful establishment of vitro models. In IFN-α treated groups,the expression of hepatitis B surface antigen( HBs Ag),hepatitis B e antigen( HBe Ag),hepatitis B core antigen( HBc Ag) and HBV-DNA in overexpression groups were significantly higher than empty vectors( P < 0. 05),while there was little difference in the IFN-λtreatment group. In addition,the expression of ISG15,Mx A,IFIT1 and p STAT1 of the empty vector were obviously higher than those in overexpression groups in the treatment group of IFN-α( P < 0. 05),but there was also no significant difference in ISGs and p STAT1 expression in IFN-λ treatment groups.USP18 inhibits the anti-HBV activity of IFN-α by suppressing the activation of the JAK/STAT signaling pathway. In contrast,USP18 has no effect on the anti-HBV activity of IFN-λ via the JAK/STAT signaling pathway.
引文
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