JAK/STAT通路在门静脉高压症脾脏促进肝卵圆细胞（肝祖细胞）恶性转化中的作用及其机制研究

Clinical data and our previous results have showed that the dysfunctional spleen in portal hypertension (PH) might facilitate the carcinogensis and development of HCC. We also found that the cytokine profiles of splenic tissue and serum from PH patients were totally different from normal control. Some of the differently expressed cytokines were related to the JAK/STAT pathway, which has been proved to play a key role in the carcinogenesis of HCC. It is generally accepted that the malignant transformation of hepatic progenitor/stem cell is the foundation for the carcinogenesis of hepatocellular carcinoma (HCC). A range of hepatic progenitor cell/oval cell (HPC/OC) has been found in HCC to be arrested in the transformational stage with neoplastic phenotype, not fully differentiated into hepatocyte. But the mechanism responsible for this phenomenon has not been elucidated yet. Here we proposed a hypothesis: "Abnormal secretion of cytokine in spleen→JAK/STAT Activation→HPC/OC malignant transformation→carcinogenesis of HCC" is another reason responsible for the development of HCC. In this study, we will demonstrate the role of PH spleen in the malignant transformation of HPC/OC from both in vivo and in vitro by evaluating the changes in cell proliferation and differentiation of HPC/OC from paracancerous tissue from patients and in DEN induced HCC SD rats model and HPC/OC culture model stimulated by splenic cell suspension. The potential mechanisms underlie the regulatory role of spleen will be elucidated by the changes in the status of JAK/STAT pathway. Furthermore, we will validate the results in cancer stem cell from HCC patients. Based on the results, we want to clarify the carcinogensis of HCC from a new opinion and provide new thoughts and clues for the prevention of HCC and clinical path for patients with cirrhosis related PH.

临床实践发现门脉高压症（PH）患者切脾后肝细胞癌（HCC）发生率下降，我们既往的研究结果显示PH患者脾血清能刺激肝癌细胞增殖，表明PH脾脏促进了HCC的发生发展，但机制不清。我们还发现PH脾脏组织及外周血细胞因子表达明显异常，其中的部分因子与肝癌中异常活化的JAK/STAT通路相关。肝修复再生过程中肝祖细胞/卵圆细胞（HPC/OC）恶性转化是HCC发生的细胞学基础。基于以上，我们首次提出"脾脏分泌细胞因子异常→JAK/STAT通路活化→HPC/OC恶性转化→肝癌发生"可能是PH脾脏促进HCC发生的重要机制之一。本项目拟以HCC患者癌旁组织中HPC为对象，采用RNA-Seq、磷酸化蛋白芯片等多种现代生物学方法，对这一假设进行验证，并对PH患者脾血中细胞因子进行初步筛选，旨在阐明PH脾对HCC发生的作用及其分子机制，从另一新角度解释HCC发生，为HCC预防及肝硬化PH患者治疗策略的确定提供新思路。

临床研究显示肝硬化门静脉高压症（Portal Hypertension，PH患者脾切除术后肝癌发生率降低，但其具体机理不明。本课题组前期实验发现PH患者脾血清能促进肝癌细胞增殖，上调原癌基因表达。我们还发现脾亢后脾组织和外周血中细胞因子表达与正常对照相比存在显著差异，其中部分细胞因子与JAK/STAT通路活化关系密切，而JAK/STATs异常活化与肝癌发生发展关系密切。因此，脾亢脾可通过多种途径影响肝癌的发生发展。目前肝癌发生被认为起源于肝再生过程中肝干/祖细胞激活发生恶性转化，基于此，我们提出异常分泌的脾源性细胞因子通过激活JAK/STATs信号通路促进肝干细胞的恶性转化。利用PH脾血清干预胎肝干细胞并以正常人外周血作对照，我们发现脾血清组胎肝干细胞胞质内出现大量颗粒物和空泡，细胞增殖加快、克隆球形成能力增强，PAS糖原染色和油红O染色显示细胞内糖原含量减少，脂肪增多，PCR检测证实细胞成分化受阻状态，表现为白蛋白表达下降、甲胎蛋白表达上升，角蛋白19表达变化不大，此外，western blotting 检测JAK2/STAT3通路在脾血清组明显活化。综上所述，脾血清影响了胎肝干细胞增殖、代谢、分化，而JAK2/STAT3可能参与了这一过程。