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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

背景与目的：已有研究表明慢性丙型肝炎患者的树突状细胞(DC)具有较低的同种刺激功能。然而，这种现象的确切机制还没有得到很好的解释。为了研究对DC细胞功能影响最大的丙型肝炎病毒多聚蛋白的组成，我们检测了7个丙型肝炎病毒蛋白基因(g-core、g-NS2、g-NS3、g-NS4A、g-NS4B、g-NS5A和g-NS5B)和5种重组蛋白(p-core、p-NS3、p-NS4、p-NS5A和p-NS5B)对DC表面标志表达及其功能的调节作用。第5天，收集细胞，用7个丙型肝炎病毒蛋白质粒用脂质体包裹，或用5个重组丙型肝炎病毒蛋白孵育。培养48h后，用流式细胞仪检测细胞表面标志物的表达。对于细胞因子…进一步分析和增殖T细胞反应，未成熟的DC和内毒素成熟的DC与AUTO的CD4+T细胞和PPD 0.1微克/孔培养。继续培养2天后，测定上清液中细胞因子的浓度。继续培养3d后，测定细胞对~3H-胸腺嘧啶核苷的摄取量。结果：NS4A、NS4B基因及NS4蛋白均可降低CD86的表达。NS4B和NS5A基因以及NS4蛋白诱导相对较低的T细胞应答。培养上清液中细胞因子检测结果显示，转导g-NS4和加入p-NS4的DC和CD4T细胞共培养上清液中Th1细胞因子浓度较低。结论：丙型肝炎病毒NS4蛋白可降低DC的CD86表达水平，降低DC功能，阻碍Th1免疫应答。然而，当区议会成熟时，这种作用可以恢复。较少