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抑制素对HCV复制的影响
Effect of prohibitin on hepatitis C virus replication
文章发布日期:2016年08月08日  来源:  作者:石娟娟,杨宁,张欣,等  点击次数:1158次  下载次数:239次

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【摘要】:目的研究抑制素(PHB)对HCV复制的影响。方法全长基因组HCV RNA体外转染人肝癌细胞系Huh7.5,构建HCV全基因组细胞模型,分别收集24、48、72、96 h培养上清液检测HCV 拷贝数;间接免疫荧光实验检测HCV核心蛋白的表达;透射电镜观察HCV感染细胞Huh 7.5-HCV的超微结构改变,鉴定HCV全基因组细胞模型。采用实时定量PCR、Western Blotting方法研究PHB在Huh 7.5-HCV细胞中的表达情况。应用RNA干扰技术检测PHB对HCV RNA的复制情况。两组间比较采用t检验。结果鉴定结果显示,成功构建HCV全基因组细胞模型。HCV RNA转染Huh7.5细胞24和48 h后,PHB mRNA表达水平分别是13.41±1.35和16.45±1.76,与对照组(101±057和101±087)相比差异均有统计学意义(t值分别为29540、31361,P值均<001);PHB蛋白表达水平亦显著高于对照组(P值均<0.01)。PHB的RNA干扰质粒(shRNA-PHB)转染Huh7.5-HCV细胞24和48h后,HCV RNA的水平分别为64.32±5.49和84.45±7.06,显著高于对照组(shRNA-control组,10.52±1.57和16.34±297,t值分别为29538、25908,P值均<0.01)。结论Huh7.5-HCV细胞中PHB表达的升高可能与HCV RNA的感染有关,PHB蛋白对HCV RNA复制有一定的抑制作用。
【Abstract】:ObjectiveTo investigate the effect of prohibitin (PHB) on hepatitis C virus (HCV) replication. MethodsHuman hepatocarcinoma Huh-7.5 cells were transfected with the full-length genome HCV RNA in vitro to establish the full-length genome cell model of HCV, and the supernatants were collected 24, 48, 72, and 96 hours later to measure HCV copies. The indirect immunofluorescence assay was used to measure the expression of HCV core proteins, and a transmission electron microscope was used to observe the changes in the ultrastructure of HCV-infected Huh-7.5 cells and identify the full-length genome cell model of HCV. Quantitative real-time PCR and Western Blot were used to measure the expression of PHB in HCV-infected Huh-7.5 cells, and the RNA interference technique was used to measure the replication of HCV RNA. The t-test was used for comparison between groups. ResultsThe results of identification showed that the HCV full-length genome cell model was successfully established. At 24 and 48 hours after Huh-7.5 cells were transfected with HCV RNA, the mRNA expression level of PHB was 13.41±1.35 and 16.45±1.76, respectively, which differed significantly from that in the control group (101±057 and 101±087,t=29540 and 31361,both P<0.01), and the 24- and 48-hour transfection groups showed a significantly higher expression level of PHB than the control group (both P<0.01). At 24 and 48 hours after Huh-7.5 cells were transfected with the RNA interference plasmids of PHB, the level of HCV RNA was 64.32±5.49 and 84.45±7.06, respectively, significantly higher than that in the control group (shRNA-control group; 10.52±1.57 and 16.34±2.97, t=29538 and 25908,all P<0.01). ConclusionIn HCV-infected Huh-7.5 cells, the increased expression of PHB is closely related to HCV RNA infection, and to a certain extent, PHB can inhibit the replication of HCV RNA.
【关键字】:肝炎病毒属; 细胞系,肿瘤; 抑制素类; RNA干扰
【Key words】:hepacivirus; cell line, tumor; inhibins; RNA interference
【引证本文】:石娟娟, 杨宁, 张欣, 等. 抑制素对HCV复制的影响[J]. 临床肝胆病杂志, 2016, 32(9): 1729-1733.

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