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目的通过对丙型肝炎病毒-原发性肝细胞肝癌(HCV-HCC)组织及细胞中精氨酸酶Ⅱ(Arg-Ⅱ)的表达进行分析,并以RNA干扰技术阻断其在细胞内的表达,初步研究Arg-Ⅱ表达在HCV-HCC发病中的意义。方法首先以基因转染技术分别将HCV基因组和对照质粒导入HuH7细胞中,以逆转录-实时荧光PCR检测HCV基因组的转录并作细胞生长曲线测定,以Western blotting技术研究HCV基因组转染对细胞内Arg-Ⅱ表达的影响,以免疫组化技术分析HCV-HCC组织中Arg-Ⅱ的表达水平,运用RNA干扰技术对Arg-Ⅱ作初步的功能分析。结果以基因转染技术分别将HCV基因组和对照质粒导入HuH7,成功建立了HuH7-HCV和HuH7-vector两个细胞系,逆转录-实时荧光PCR检测表明HuH7-HCV细胞的HCV转录水平为0.5~5拷贝/细胞。Western blotting结果显示,与对照细胞相比,HuH-7-HCV细胞的Arg-Ⅱ含量升高3.9倍。免疫组化技术分析HCV-HCC组织标本中Arg-Ⅱ的表达,结果呈强阳性,而对照标本中基本不表达。采用RNA干扰方法阻断HuH7-HCV细胞的Arg-Ⅱ表达,发现细胞增殖被明显抑制。结论Arg-Ⅱ在HCV阳性肝细胞系中及HCV-HCC组织中异常表达并可能参与HCV-HCC的发病机制。
Objective To analyze the expression of arginase Ⅱ (Arg-Ⅱ) in the tissues and cells of hepatitis C virus-hepatocellular carcinoma (HCC) and to block its intracellular Expression and Preliminary Study of the Significance of Arg-Ⅱ Expression in the Pathogenesis of HCV-HCC. Methods HCV genome and control plasmid were respectively transfected into HuH7 cells by gene transfection technique. The transcription of HCV genome was detected by RT-PCR and the cell growth curve was determined. Western blotting was used to study the effect of HCV genome transfection on intracellular Arg-Ⅱ expression, immunohistochemical analysis of HCV-HCC tissue Arg-Ⅱ expression levels, the use of RNA interference technology Arg-Ⅱ for preliminary functional analysis. Results Huh7-HCV and HuH7-vector cell lines were successfully transfected with HCV genome and control plasmid respectively by gene transfection. Reverse transcriptase-real-time PCR showed that the HCV transcript level of HuH7-HCV cells was 0.5 ~ 5 copies / cell. Western blotting showed that Arg-II content in HuH-7-HCV cells increased by 3.9-fold compared with control cells. Immunohistochemical analysis of HCV-HCC tissue samples Arg-Ⅱ expression, the result was strongly positive, while the control specimens were basically not expressed. The Arg-Ⅱ expression of HuH7-HCV cells was blocked by RNA interference, and cell proliferation was significantly inhibited. Conclusion Arg-Ⅱ is abnormally expressed in HCV-positive hepatocytes and in HCV-HCC tissues and may be involved in the pathogenesis of HCV-HCC.