HBx介导肝卵圆细胞通过EMT导致肝癌复发和转移的机制研究

At present the study of HCC recurrence and metastasis is less. Our previous studies have shown that transfection of hepatitis B virus X gene (HBx) of the liver oval cells grown in nude mice, fed aflatoxin (AFB1) can form liver cancer sarcoma. Research found in the absence of transforming growth factor- β (TGF-β), only activated oval cells is not sufficient to cause liver cancer formation, and TGF-β is a key factor to induce epithelial - mesenchymal transition (EMT), our previous study also confirmed oval cells may induce EMT in the microenvironment. Based on the above studies we hypothesize: HCC recurrence and metastasis may be caused by oval cells activated and the occurrence of EMT, due to the role of HBx, oval cells mutation generated hepatoma cells, leading to HCC recurrence and metastasis. In this study, through the oval cell lines, nude mice and clinical HCC patients, using transfection and gene knockout technologies, through in vivo and in vitro studies to clarify the role of EMT and the molecular mechanism of HBx in the activated oval cells for HCC recurrence and metastasis, and this study will provide direct experimental evidence that HCC recurrence and metastasis are derived from oval cells, so as to provide theoretical support for HCC clinical biology search with more effective treatment.

肝癌的复发和转移当前研究较少。我们前期研究显示转染乙肝病毒X基因（HBx）的卵圆细胞种植于裸鼠肝内，加喂黄曲霉毒素（AFB1）可形成肝癌肉瘤，研究发现在缺乏转化生长因子β（TGF-β）的情况下激活卵圆细胞不足以引发肝癌形成，而TGF-β又是诱导上皮-间质转化（EMT）的关键因子，我们前期研究证实卵圆细胞在微环境作用下可发生EMT，在此基础上我们提出假设：肝癌的复发和转移可能是卵圆细胞被激活并发生EMT，由于HBx的作用，卵圆细胞变异生成肝癌细胞，从而导致肝癌的复发和转移。本研究将通过卵圆细胞株，裸鼠和临床肝癌病人为研究对象，采用基因的转染和敲除等技术，通过体内外试验阐明EMT和HBx在卵圆细胞激活后的作用及其分子机制，为肝癌术后复发和转移是否来源于卵圆细胞提供直接的试验证据，从而为寻找更有效的肝癌生物学治疗提供理论支持。

肝癌的复发和转移可能是卵圆细胞被激活并发生EMT，由于HBx的作用，卵圆细胞变异生成肝癌细胞，从而导致肝癌的复发和转移。研究内容主要分为四个部分：1、检测复发肝癌组织和癌旁组织中肝卵圆细胞的增殖，观察、分析其与HBx以及肝癌复发和转移的关系；2、探索HBx对卵圆细胞的影响，以及发生EMT后对稳定表达HBx的卵圆细胞的影响；3、建立2-AAF/PH（2-乙酰氨基芴+2/3肝切除）大鼠卵圆细胞增殖模型，模拟人肝癌术后肝脏微环境改变，探索卵圆细胞实际发挥的作用；4、建立HBx 和TGF-β协同诱导卵圆细胞转化为肝癌的动物模型，明确肿瘤的性质，检测HBx和TGF-β信号相关分子的表达。课题研究结果表明，HBx与TGF-β可以共同刺激卵圆细胞激活MAPK信号通路，上调miR-199a-3p导致其分化，诱导其发生EMT导致其侵袭转移，为肝癌的预防和治疗提供了新的靶点。