Molecular mechanisms of chronic hepatitis C progression

慢性丙型肝炎进展的分子机制

基本信息

批准号：
15590649
负责人：
IWASAKI Yoshiaki
金额：
$ 1.92万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590649/
关键词：
hepatitis C virus HLA immune response dendritic cell 抗原特異的刺激 C型肝炎 HCV プロテアゾーム

项目摘要

Background and Aims : It has been reported that dendritic cells (DC) of chronic hepatitis C patients showed reduced allo-stimulatory function. However, the exact mechanisms of this phenomenon are not well defined. To investigate the component of HCV polyprotein that has the most important effect on DC cell function, we examined the modulatory effects of 7 HCV protein genes (g-core, g-NS2, g-NS3, g-NS4A, g-NS4B, g-NS5A, and g-NS5B) and 5 recombinant proteins (p-core, p-NS3, p-NS4, p-NS5A, and p-NS5B) on the DC surface marker expression and its functions.Methods : Peripheral blood mononuclear cells (PBMC) were separated from healthy volunteers using a Ficoll gradient, and CD14+ cells were enriched using CD14 microbeads. On day 5, the cells were harvested and pulsed with 7 HCV protein plasmids by the lipofection method or incubated with 5 recombinant HCV proteins. After culturing for 48 hrs, the cells were assayed for cell surface marker expression by flow cytometric analysis. For cytokin … More e analysis and proliferative T cell response analysis, the immature DC and LPS matured DC were cultured with auto CD4+ T cells and PPD 0.1 microgram / well. After 2 more days of culture, the supernatants were measured for cytokine concentration. After a further 3 days of culture, 3H-thymidine uptake was measured. The results were all shown as the ratio of HCV protein data to data of the control vector or buffer control.Results : The NS4A and NS4B genes and NS4 protein reduced the expression of CD86. The NS4B and NS5A genes and NS4 protein induced relatively low T cell responses. Cytokines of the culture supernatant showed that the concentration of Th1 cytokines of the supernatants were low in g-NS4 transduced and p-NS4 - added DC and CD4 T cell cocultures. However, these effects of HCV proteins on DC function were all restored by LPS maturation.Conclusion : HCV NS4 protein may reduce the CD86 expression levels and the function of DC and hamper the Th1 immune responses. However, this effect can be restored when the DCs were well matured. Less

背景和目的：据报道，慢性丙型肝炎患者的树突状细胞（DC）表现出降低的同种刺激功能。但是，这种现象的确切机制尚未很好地定义。为了研究对DC细胞功能具有最重要作用的HCV多蛋白的成分，我们检查了7种HCV蛋白基因（G-CORE，G-NS2，G-NS3，G-NS3，G-NS4A，G-NS4A，G-NS4B，G-NS5A和G-NS5A和G-NS5B和G-NS5B）和5个重组蛋白（P-NS5，P-NS3，P-NS3，P-N，P-NS3，P-N，P-NS3，P-NS3，P-NS3，P-NS4，P-NS4，P-NS3，P-NS4，P-NS3，G-NS4A，G-NS4B，G-NS5A和P）的调节作用。 DC表面标记表达及其功能上的P-NS5B）方法：使用Ficoll梯度从健康志愿者分离外周血单核细胞（PBMC），并使用CD14微粒富含CD14+细胞。在第5天，通过LiPofection方法收集细胞并用7 HCV蛋白质粒脉冲，或与5种重组HCV蛋白一起孵育。聚集48小时后，通过流式细胞仪分析将细胞分配用于细胞表面标记表达。对于Cytokin…更多的E分析和增殖T细胞反应分析，将未成熟的DC和LPS成熟DC用自动CD4+ T细胞和PPD 0.1微克 /孔培养。在培养2天后，测量上清液的细胞因子浓度。在培养3天后，测量了3H-胸腺苷的摄取。结果均显示为HCV蛋白数据与控制载体或缓冲区控制的数据的比率。分子：NS4A和NS4B基因和NS4蛋白降低了CD86的表达。 NS4B和NS5A基因和NS4蛋白诱导了相对较低的T细胞反应。培养上清液的细胞因子表明，上清液的Th1细胞因子的浓度在G-NS4转导的G-NS4转导和p-NS4-添加的DC和CD4 T细胞共培养物中。但是，HCV蛋白对DC功能的这些影响均通过LPS的成熟恢复。结论：HCV NS4蛋白可以降低CD86表达水平以及DC和DC的功能，并妨碍TH1免疫调查剂。但是，当DC良好成熟时，可以恢复这种效果。较少的