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Identification of the key molecules for hepatitis C virus (HCV) replication and the translation research for the noble protocols against HCV using cell-based HCV replication model.

使用基于细胞的 HCV 复制模型鉴定丙型肝炎病毒 (HCV) 复制的关键分子以及针对 HCV 的高贵方案的翻译研究。

基本信息

批准号：
17590650
负责人：
HIASA Yoichi
金额：
$ 2.18万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590650/
关键词：
Hepatitis C virus HCV replication model Protein kinase R Interferon Ribavirin Interleukin-8 T cell anti-HCV therapy 抗HCV治療 EVR

项目摘要

We established the cell-based hepatitis C virus (HCV) replication system with recombinant adenovirus vectors and with T7 stable cell lines. With pH77 and Ad-T7pol, HCV positive and negative strand RNA expression was strongest in the first 2 to 3 days post-infection and diminished thereafter, but were expressed throughout the 9 days. This sustained expression of HCV-RNA was also seen in the system with T7 stable cell lines. In these systems, interferon (IFN) inhibits HCV replication, and upregulates the protein kinase R (PKR) pathway.IFN and ribavirin (RBV) combination therapy is the current standard of antiviral therapy for chronic HCV. Among IFN-induced proteins, PKR is considered as a key molecule to eliminate HCV. On the other hand, interleukin-8 (IL-8) has been reported to inhibit IFN-induced antiviral responses. In our replication model, both HCV expression and IFN, induced PKR and its regulated proteins effectively. HCV protein expression in turn was directly regulated by PKR. From our experiments, PKR is directly antiviral for HCV, and is a central mediator of IFN' s effects against HCV. About IL-8, it was induced by both IFN and RBV in our systems. We speculate that IL-8 induction by both agents might not produce sufficiently antiviral effects. Rather, increased IL-8 might have an indirect role as an immunomodulator or as a chemokine in recruitment of cells that eliminate HCV.Additionally, we did in vivo assay by collecting peripheral blood T cells from the chronic hepatitis C patients who were treated by IFN and RBV combination therapy. In the patients with early viral response (EVR), HCV-RNA become undetectable within 12 weeks, PKR mRNA in T cells are significantly higher than that in the patients with non-EVR.From these results, we concluded that PKR and IL-8 will be key molecules for HCV replication as well as HCV persistence. Moreover, we have to consider to establish the protocol which can upregulate PKR effectively for the stronger anti-HCV therapy.

我们用重组腺病毒载体和T7稳定细胞系建立了以细胞为基础的丙型肝炎病毒（HCV）复制系统。在pH 77和Ad-T7 pol条件下，HCV正、负链RNA在感染后2 ~ 3d表达最强，此后逐渐减弱，但在感染后9d内均有表达。在具有T7稳定细胞系的系统中也观察到HCV-RNA的这种持续表达。在这些系统中，干扰素（IFN）抑制HCV复制，并上调蛋白激酶R（PKR）途径，IFN和利巴韦林（RBV）联合治疗是目前慢性HCV抗病毒治疗的标准。在IFN诱导的蛋白质中，PKR被认为是消除HCV的关键分子。另一方面，已报道白细胞介素-8（IL-8）抑制IFN诱导的抗病毒应答。在我们的复制模型中，HCV表达和IFN都有效地诱导PKR及其调节蛋白。HCV蛋白表达反过来又受PKR的直接调控。根据我们的实验，PKR对HCV具有直接抗病毒作用，并且是IFN抗HCV作用的中心介质。IL-8在IFN和RBV的共同诱导下产生。我们推测这两种药物诱导IL-8可能不会产生足够的抗病毒作用。相反，增加IL-8可能有一个间接的作用，作为一种免疫调节剂或作为一种趋化因子，在招聘的细胞，消除HCV.Additionally，我们做了体内试验，收集外周血T细胞从慢性丙型肝炎患者谁是治疗IFN和RBV联合治疗。在早期病毒应答（EVR）患者中，HCV-RNA在12周内检测不到，PKR mRNA在T细胞中的表达明显高于非EVR患者，提示PKR和IL-8可能是HCV复制和持续存在的关键分子。此外，我们必须考虑建立有效上调PKR的方案，以更强的抗HCV治疗。