中文摘要：

目的 在乙型肝炎病毒（HBV）相关性肝细胞肝癌（hepatocellular carcinoma,HCC）中探讨乙肝病毒x蛋白（HBx）对骨桥蛋白（osteopontin,OPN）表达及肝癌细胞干性的影响。方法 采用免疫印迹（Western blot,WB）及免疫组化等方法在肝癌组织标本上检测OPN表达水平与患者性别、年龄、AFP、HBV病毒复制量、肿瘤大小、TNM分期等临床病理因素的关系;在稳定整合HBV基因组的HepG2.2.15细胞系及慢病毒介导的HBx转染HepG2细胞中,进一步验证HBV及HBx过表达对OPN及干性相关转录因子Nanog、Oct4,干性细胞表面标记分子CD44、CD90、Ep CAM表达水平的影响。结果 免疫组化及WB检测结果均表明OPN在HBV阳性的HCC肝癌组织中的表达水平显著高于HBV阴性的肝癌组织（P〈0.05）。HepG2.2.15细胞中OPN的mRNA及蛋白质含量较HepG2细胞显著增加（P〈0.05）;在HepG2细胞中,过表达HBx显著上调OPN mRNA及蛋白质水平（P〈0.05）,流式细胞术定量分析显示过表达HBx的HepG2细胞中Ep CAM、CD90阳性的干性细胞数量明显升高（P〈0.05）,实时定量PCR显示干性相关转录因子Nanog、Oct4及其表面标志分子CD44、CD90、Ep CAM水平显著升高。结论 HBx能够显著上调OPN的表达,并增强肝癌细胞的干性,提示HBx/OPN/肝癌细胞干性增强通路可能在HBV相关HCC患者预后不良中具有重要作用。

英文摘要：

This study aimed to investigate the effect of Hepatitis B virus X protein（HBx） on osteopontin（OPN） expression and the stem cell-like properties of hepatocellular carcinoma（HCC）. The expression of OPN wasmeasured by immunohistochemistry and Western blotting in both HBV-positive and-negative HCC liver tissues,and its correlation with clinical pathological factors including age, sex, AFP, HBV infection, tumor size, TNM stagewere analyzed. HepG2.2.15, a cell line with stably integrated HBV genome, and HepG2 cells expressing hepatitis Bvirus proteins were used to detect the effects of HBV infection on OPN expression. Furthermore, the recombinantlentivirus expressing HBx was used to infect HepG2 cells, and the effects of HBx on the stemness markers（CD44,CD90, Ep CAM）, transcriptional regulators（Nanog, Oct4） and OPN gene expression were tested. Data showed thatOPN expression in HBV-positive tissues wassignificantly higher than that in HBV-negative HCCsample tissues（P〈0.05）. Accordingly, OPN mRNAand protein levels were significantly higher inHepG2.2.15 cells compared to HepG2 cells（P〈0.05）.Ectopic overexpression of HBx significantly increased the stem-like cell numbers with both EpCAM^＋and CD90^＋markers. As a further support, enforced HBx expression inHepG2 cells upregulated OPN levels as well as the mRNA levels of CD44, CD90, EpCAM, Nanog and Oct4. Takentogether, HBx upregulates OPN promoting the stem cell-like proterties of tumor cells in hepatocarcinoma. Theelucidation of HBx/OPN/stemness pathway in HCC help to better understand the molecular mechanism ofhepatocarcinogenesis, particularly, under chronic HBV infection.