低氧诱导的上皮间质化转变在肝细胞性肝癌中诱导肿瘤微环境免疫抑制的机制研究

发布时间：2018-08-20 14:29

【摘要】：目的：门静脉癌栓(PVTT)的肝癌病人预后较差,可能与其转移相关,但是其具体机制尚未明确。本文目的为了进一步明确有PVTT的肝癌病人的HIF-1α的表达情况,并分析研究其对肿瘤微环境的影响。材料与方法：回顾性分析了2007至2010年于浙江大学医学院附属第二医院行肝癌根治性切除的病人,用他们的肿瘤组织做免疫组织化学(IHC)以及定量RT-PCR检测(qRT-PCR),检测其中CCL20,HIF-1α与IDO的表达量,并根据CCL20的表达情况,分析其与患者生存预后的关系。在体外使用低氧培养箱模拟体内低氧,分析肝癌细胞系在低氧下的细胞因子的变化。为了进一步分析其低氧后诱导的细胞因子对肿瘤免疫微环境的影响,我们将单核细胞来源的巨噬细胞与肿瘤细胞系一起共培养。结果：研究发现PVTT的病人肿瘤组织中,HIF-1α明显高表达,同时我们发现HIF-1α可以显著促进肝癌细胞的CCL20的分泌,针对这一现象,我们行ChIP试验证明,在CCL20的基因启动子位点上,有低氧应答元件(Hypoxia response element,HRE)存在。同时我们在病人组织中也发现CCL20与IDO处于高表达状态,并且两者具有明显的线性关系,我们在体外实验证明单核细胞在CCL20的作用下,IDO显著高表达,分化成为具有免疫抑制作用的单核细胞来源的巨噬细胞,机制上主要是通过Statl磷酸化以及核转位。这群IDO+的巨噬细胞在体外,可以诱导Foxp3+的Treg样形成。此外我们在GEO数据库里证明,CCL20与IDO在有无血管侵犯的病人体内的表达水平不同,并且在只有在有血管侵犯的病人组内有明显的线性相关。结论：我们认为可能HIF-1α/CCL20/IDO这条轴线,在肝癌中具有重要作用,其激活能诱导肝癌细胞EMT,并进而导致肿瘤微环境中免疫抑制的形成,从而导致肝癌复发转移加速。我们认为阻断这条通路,可能可以做为肝癌治疗的靶点。
[Abstract]:Objective: the prognosis of HCC patients with portal vein tumor thrombus (PVTT) is poor, which may be related to metastasis, but the mechanism is not clear. Objective to investigate the expression of HIF-1 伪 in hepatocellular carcinoma with PVTT and its effect on tumor microenvironment. Materials and methods: patients undergoing radical hepatectomy in the second affiliated Hospital of Zhejiang University School of Medicine from 2007 to 2010 were retrospectively analyzed. Immunohistochemical (IHC) and quantitative RT-PCR (qRT-PCR) were used to detect the expression of CCL20hIF-1 伪 and IDO in their tumor tissues. According to the expression of CCL20, the relationship between the expression of CCL20HIF-1 伪 and the survival and prognosis of the patients was analyzed. Hypoxia incubator was used to simulate hypoxia in vitro to analyze the changes of cytokines in hepatoma cell lines under hypoxia. In order to further analyze the effect of cytokines induced by hypoxia on tumor immune microenvironment, macrophages derived from monocytes were co-cultured with tumor cell lines. Results: we found that the expression of HIF-1 伪 was significantly higher in the tumor tissues of patients with PVTT, and we found that HIF-1 伪 could significantly promote the secretion of CCL20 in hepatoma cells. In view of this phenomenon, we conducted ChIP test to prove that it was at the promoter site of CCL20 gene. Hypoxia response element (Hypoxia response element HRE) is present. At the same time, we also found that CCL20 and IDO were highly expressed in patient tissues, and there was a significant linear relationship between them. We demonstrated in vitro that mononuclear cells were significantly overexpression of IDO under the action of CCL20. The mechanism of differentiation into macrophages derived from monocytes with immunosuppressive effect is mainly through Statl phosphorylation and nuclear translocation. The macrophages of IDO can induce Treg like formation of Foxp3 in vitro. In addition, we demonstrated in the GEO database that the expression of CCL20 was different from that of IDO in patients with or without vascular invasion, and that there was a significant linear correlation only in patients with vascular invasion. Conclusion: it is suggested that the axis of HIF-1 伪 / CCL20 / IDO may play an important role in HCC, and its activation can induce the formation of immunosuppression in the tumor microenvironment and accelerate the recurrence and metastasis of HCC. We believe that blocking this pathway may be a target for the treatment of liver cancer.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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