肝癌介入栓塞后HMGB1调节Kupffer细胞与肝癌细胞的交互对话及对肝癌侵袭转移影响的分子机制研究

The local recurrence after transarterial chemoembolization (TACE) always occur at the margin of tumor, because of inflammation developed after hypoxia which lead to iodine-oil cleaning up. The hypoxia after embolization is important to Kupffer cells activation and recruiting. In our priliminary study, we identified that hypoxia could be induced after embolization, which could not only stimulate hepatocellular carcinoma (HCC) cell motility, but also induce epithelial-mesenchymal transition, resulting in HCC progression. In pre-experiment, we also found that high mobility group protein 1 (HMGB1) is secreted higher by HCC in hypoxia. HMGB1 play a role as inflammatory cytokine, which could activate Kupffer cells and involved in lots of hepatic disease, such as hepatic ischemia- reperfusion injury, hepatic fibrosis. Based on the above information, in this study, we will elucidate the cross-talk between Kupffer cell and hepatoma and its significance in HCC recurrence and metastasis after embolization. Using coculture analysis, specific chemotractant models and mice experiments for intervention, we will emphsis on the biological roles and the underly mechanisms of the HMGB1 in modulating the Kupffer cell and hepatoma, which in turn leading tumor progress in hypoxia microenvironment. In addtion, our study will also investigate the effect and the associated molecullar mechnisms of HMGB1 on HCC cell invasion and metastasis, which could be response to the activation of TLR4/NF-κB signal pathway. Modulating HMGB1 or Kupffer cell will repress the invasive potential of HCC cells, and leading to reduced HCC recurrence and improve survival.

肝癌介入栓塞后局部缺氧引起的炎症反应与转移和复发有密切关系。本课题前期研究发现缺氧诱导肝癌细胞发生上皮间质转化，预实验显示缺氧引起肝癌细胞分泌HMGB1，基于炎性因子HMGB1可以活化Kupffer细胞（KCs），参与多种肝脏疾病的发生发展过程。因此本课题提出“HMGB1介导肝癌细胞与KCs的交叉对话影响肝癌转移”的设想，拟用体外细胞共培养、趋化及干预实验，通过ELISA、Western blot和PCR等技术研究肝癌细胞在缺氧条件下释放的HMGB1对KCs的活化，以及活化的KCs分泌的HMGB1对肝癌细胞侵袭转移能力的影响，明确TLR4/NF-κB信号通路调节KCs和肝癌细胞的交叉对话的作用。另外通过肝癌动物模型，验证介入栓塞联合HMGB1抑制，对KCs活化和肝癌转移的抑制效果。以期为临床寻找抑制肝癌侵袭和转移的治疗靶点，提高肝癌介入栓塞的疗效。

肝细胞肝癌（HCC）介入栓塞后，肿瘤微环境发生一系列变化能引起肝癌的存活、复发和转移，其中局部缺氧引起的炎症反应与肿瘤的转移和复发有密切关系，但其机制尚不明确。高迁移率组蛋白-1（HMGB1）作为一个内源性炎性介质，在细胞的增殖、分化和运动迁移中发挥着重要作用，同时与包括肝癌在内的多种肿瘤预后不良密切相关。本研究发现，缺氧条件下随着HIF-1a的升高，HCC中HMGB1的表达升高，HIF-1a下调后HMGB1表达受抑制。结果提示HIF-1a在缺氧诱导的HCC细胞HMGB1表达升高中起关键作用。通过共培养模型，我们发现，缺氧条件下，HCC分泌的HMGB1能促使巨噬细胞活化，巨噬细胞浸润能力增加，巨噬细胞分泌的TNF-a，IL-6，IL-1b和CCL2表达水平升高。由此说明HCC细胞释放的HMGB1是介导缺氧条件下巨噬细胞向肿瘤浸润的重要因素。进一步研究发现巨噬细胞来源的IL-6能活化HCC细胞STAT3信号传导通路，促进HCC细胞发生上皮间质转化（EMT），进而促进HCC侵袭迁移能力增加。利用IL-6受体抗体干预后，缺氧条件下HCC细胞的EMT相关基因表达的影响被有效地抑制，同时肿瘤细胞侵袭迁移能力明显减低。动物实验显示HMGB1抑制后，肿瘤浸润巨噬细胞明显减少，IL-6表达降低和STAT3信号传导活化减少，明显抑制HCC细胞EMT和远处转移。本项目提示肝癌介入栓塞后，缺氧微环境中肿瘤表达的HMGB1能促进巨噬细胞活化，巨噬细胞活化后分泌的IL-6能通过IL6—STAT3信号传导通路，引起肝癌细胞发生EMT，能促进残癌存活及复发转移。本研究明确了HMGB1调节巨噬细胞与肝癌细胞交互对话促进肝癌侵袭转移的作用，同时发现深入探索HMGB1准确、高效的输送形式和作用方式，切断肿瘤细胞与巨噬细胞交互对话的环路，可能是抑制肿瘤复发转移的一个有效解决途径。本研究为我们深入开展后续研究拓展了思路和方向，对明确肿瘤微环境对肿瘤复发转移的影响、提高肝癌治疗的疗效具有重要的意义。