肝癌发生中脂多糖参与肝前体细胞异常激活和分化的机制研究

The abnormal activation and differentiation of the HPC compartment is associated with tumor development and progression in hepatocellular carcinoma (HCC). It is well established that lipopolysaccharides (LPS) plays an important role in liver cancer microenvironment. Our previous findings demonstrated that LPS regulated the stemness of TLR4 positive HCC cells for tumor initiation. We found that LPS promoted hepatic progenitor cells (HPC) activation and sustained stemnesss of HPC for the tissue repair in chronic liver injury. However, long term treatment of LPS enhanced the tumorogenisis of HPC. We speculated that LPS might induce the abnormal activation and differentiation of HPC for the occurence of HCC. Our task was designed to investigate the effects of LPS on the tumor development and HPCs activation, as well as the tumorigenic properties of HPC in animal models for primary HCC. The stemness of HPCs and abnormal differentiation was observed under LPS condition both in vivo and in vitro. The downstream of TLR4 signaling and HPC activation and differentiation related signaling pathway were analyzed by PCR microarray and gene expression profile to screen the critical molecule, which determined the activation and differentiation of HPCs. The function of this critical molecule was validated using negative regulation methods to clarify the mechanism of HPC abnormal activation and differentiation under LPS treatment. The clinical and follow up data were analyzed retrospectively to evaluate the correlation between the specific molecule expression and prognosis of HCC patients. These findings could provide the theoretical basis for clinical HCC diagnosis and treatment and improve our understanding of the mechanisms underlying HCC development and progression.

肝癌发生与肝前体细胞（HPCs）异常激活和分化有关，脂多糖（LPS）是肝癌微环境的重要组成部分。我实验室前期研究发现LPS参与TLR4阳性肝癌细胞的干性调控，在肝癌发生中发挥重要作用；慢性肝损伤中LPS通过TLR4促进HPCs激活和干性维持参与损伤修复，但LPS长期刺激则导致HPCs成瘤性增加。推测肝癌发生可能与LPS诱导HPCs异常激活和分化有关。本课题拟在原发性肝癌模型中观察LPS对肝癌发生、HPCs激活及成瘤能力的影响；探讨LPS对HPCs干性特征及异常分化的影响；利用芯片技术围绕TLR4下游及HPCs激活和分化调控相关通路筛选介导LPS参与HPCs异常激活和分化的关键分子，并通过反向调控验证关键分子的功能，阐明LPS参与肝前体细胞异常激活和分化的机制；利用临床标本和随访资料，分析相关因子表达与患者预后相关性。研究结果有助于认识肝癌发生的机制，为临床诊疗提供了新思路和理论依据。

肝前体细胞（hepatic progenitor cells，HPCs）在肝癌炎症微环境的调控下被激活，从而分化成为成熟的肝细胞，胆管细胞，促进肝脏的损伤修复，或者异常分化为肿瘤起始细胞，参与肝癌的发生。脂多糖（Lipopolysaccharide，LPS）是肝癌炎症微环境的重要组成部分。我们研究发现LPS可诱导HPCs向肌成纤维细胞分化，HPCs来源的肌成纤维细胞参与肿瘤发生微环境的形成，进而促进HPCs的异常分化从而形成肝癌。HPCs源性的肌成纤维细胞可能通过分泌IL-6和TNF-α促进正常HPCs增殖及异常分化。TNF-α是脂多糖下游重要的炎症因子，我们深入探讨了TNF-α对HPCs功能的影响。研究发现TNF-α通过结合其受体TNF-R2，激活下游STAT3信号通路，促进HPCs的激活，提示TNF-α可能在HPCs的激活中发挥非常重要的作用，同时TNF-α可能也在肝脏慢性炎症损伤修复中发挥重要作用。肝癌通常发生在慢性肝损伤的晚期，抑瘤素M (oncostatin M, OSM) 是一种肿瘤相关细胞因子，在肝硬化和肝癌患者中均高度表达。我们研究发现OSM促进体内HPCs活化，但在体外实验中对 HPCs细胞的增殖无影响。此外，OSM诱导TNF-α的分泌和 CD68阳性巨噬细胞的累积，均与HPCs激活呈正相关。总之，OSM通过调节肝脏炎症微环境，在肝癌发生中起重要作用。综上所述，我们不仅阐明了LPS诱导HPCs异常分化的作用和机制，更进一步深入研究了LPS下游炎症因子TNF-α对HPCs激活的影响，以及炎症因子OSM对HPCs活化的影响，在完成本课题研究内容的基础上，深入研究了影响HPCs激活和活化的相关因素，进一步认识了HPCs促进肝癌发生的机制，为靶向HPCs预防肝癌发生提供了理论依据。