基于CCL20-CCR6信号轴探讨中药QHF复方抗肝癌免疫逃逸作用及分子机制

The immune escape of hepatocellular carcinoma (HCC) is closely related to regulatory T cell (Treg), and the reduction of Treg cell number and its migration to tumor tissue can significantly inhibit the immune escape of HCC. QHF compound which we have screened out for the treatment of liver cancer before showed that it can inhibit the number of liver cancer Treg cells and Treg cell-specific markers FOXP3 and CCL20, CCR6 expression in cancer tissues. Therefore, CCL20 cDNA transfection technique is used to construct CCL20 high expression hepatocellular carcinoma cell lines and mouse hepatocellular carcinoma model; and FCM, RT-PCR, Western Blot, IHC and other techniques are used to further detect the effect of QHF compound and its components on the expression of CCR6 protein and gene in CCL20 and Tregs in cancer tissues, and to explore the action targets and the molecular mechanisms of QHF compound and its components inhibit the migration of Treg cells to the tumor through CCL20 / CCR6 signal axis, which will look for an effective anti-liver cancer immune drugs and therapeutic targets to provide experimental evidence.

肝细胞肝癌（hepatocellular carcinoma，HCC）免疫逃逸与调节性T 细胞（Regulatory T cell, Treg）密切相关，减少Treg细胞数量及其向肿瘤组织迁移可显著抑制HCC免疫逃逸。我们前期筛选得到的治疗肝癌中药验方QHF复方，初步显示其有抑制肝癌小鼠外周Treg细胞数量和癌组织内Treg细胞特异性标志物FOXP3以及CCL20、CCR6表达的作用。鉴于此，本项目拟通过CCL20 cDNA转染技术，构建CCL20高表达肝癌细胞株和小鼠肝癌模型，采用FCM、RT-PCR、Western Blot、IHC等技术进一步验证QHF复方及各组分对癌组织中CCL20和Treg中 CCR6蛋白和基因表达等的影响，并探讨QHF复方及各组分通过CCL20/CCR6信号轴抑制Treg细胞向肿瘤迁移的分子机制和作用靶点，为寻找有效的抗肝癌免疫药物和治疗靶点提供实验依据。

肝细胞肝癌（hepatocellular carcinoma，HCC）免疫逃逸与调节性T细胞（Regulatory cells, Tregs）密切相关，减少Treg细胞数量及其向肿瘤组织迁移可显著抑制HCC免疫逃逸。课题组前期筛选得到的治疗肝癌中药验方QHF复方，初步显示其有抑制肝癌小鼠外周Treg细胞数量和癌组织内Treg细胞特异性标志物FOXP3以及CCL20、CCR6表达的作用，但具体分子机制未明确。本项目构建并筛选小鼠脾淋巴细胞和肝癌细胞株（H22、Hepa1-6）最佳共培养体系，利用CCL20 cDNA转染技术成功构建CCL20高表达肝癌细胞株和小鼠肝癌模型，借助流式细胞术、免疫荧光双染、Westren Blot等现有分子技术多方面阐明了QHF复方通过CCL20/CCR6信号轴抑制Treg细胞向肝肿瘤迁移的分子机制。研究发现脾淋巴细胞和肝癌细胞（H22、HepG2）的最佳浓度比分别为10:1，测定24h为最佳共培养时间，不同浓度的QHF均能明显促进淋巴细胞以及共培养后的淋巴细胞的增殖和共培养体系淋巴细胞FOXP3、CCR6基因表达和肝癌细胞CCL20表达。研究还发现QHF复方能抑制肝癌荷瘤小鼠和高转染CCL20小鼠的肿瘤生长、肿瘤和脾脏组织的CCL20+Foxp3+表达以及外周血IL-2、IFN-γ、TGF-β和IL-10的含量，同时能抑制CCL20和CCR6的基因、蛋白表达。阐明中药QHF复方及各组分抗肝癌免疫逃逸的作用是通过抑制肿瘤细胞募集Treg细胞来实现的，明确了QHF复方抑制肿瘤细胞分泌CCL20，下调Treg细胞CCR6表达的分子机制和作用靶点。此项目为该药应用于抗肝癌免疫逃逸的临床研究和寻找新的治疗靶点提供理论依据。