不同剂量X射线对cGAS⁃STING信号通路及肿瘤免疫微环境的影响

Effects of different doses of X⁃rays on cGAS⁃STING signaling pathway and tumor immune microenvironment

目的研究不同剂量X射线照射对肝癌细胞免疫微环境及cGAS⁃STING信号通路的影响。方法C57BL/C小鼠右侧腋部皮下注射Hepa 1-6肝癌细胞,建立皮下成瘤肝癌模型,按顺序随机分为0、4、8、12 Gy照射组,每组10只,监测小鼠体重和肿瘤体积。照射后28 d收集标本,采用ELISA法和流式细胞术,比较各组肿瘤组织内巨噬细胞肿瘤坏死因子α(TNF⁃α)、干扰素γ(IFN⁃γ)、白介素6(IL⁃6)、趋化因子配体5(CCL5)、IL⁃10、IL⁃13、转化生长因子β(TGF⁃β)、IL⁃4和巨噬细胞M1、M2表型比例(M1/M2)的变化。采用实时荧光定量聚合酶链式反应(qRT⁃PCR)和免疫印迹实验检测肝癌细胞cGAS⁃STING信号通路相关基因与蛋白表达情况。结果随着照射剂量的增加,肿瘤体积明显减小(F=8.42,P<0.05),细胞坏死比例增加(F=3.89,P<0.05),除IL⁃4之外的巨噬细胞相关细胞因子含量增加(F=6.32~15.50,P<0.05),肝癌肿瘤免疫微环境中的M1、M2型巨噬细胞的比例升高(F=5.46、5.14,P<005)。肝癌细胞内的cGAS⁃STING信号通路基因表达与蛋白表达水平升高(mRNA:F=6.35、16.10,P<0.05;蛋白:F=71.31、37.15,P<0.05)。结论X射线照射可激活肝癌细胞的cGAS⁃STING信号通路,促使肿瘤免疫微环境重塑。

Objective To study the effects of different doses of X-ray irradiation on the immune microenvironment and cGAS-STING signaling pathway of hepatocellular carcinoma cells.Methods C57BL/C mice were subcutaneously injected with Hepa 1-6 hepatocellular carcinoma cells in the right axilla to establish a subcutaneous tumor-forming hepatocellular carcinoma model.The mice were randomly divided into 0,4,8,12 Gy irradiation groups,with 10 mice in each group.The body weights and tumor volumes were monitored.Specimens were collected 28 d after irradiation.The ELLSA and Flow Cytometry method was used to compare the macrophage-associated cytokines tumor necrosis factor-α(TNF-α),interferon-(IFN-),interleukin-6(IL-6),chemokine ligand 5(CCL5),IL-10,IL-13,transforming growth factor-β(TGF-β),IL-4 and macrophage M1,M2 phenotype ratio(M1/M2).Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and immunoblotting assay were used to detect the expression of genes and proteins related to the cGAS-STING signaling pathway in hepatoma cells.Results With the increase of irradiation dose,the tumor volume was significantly reduced(F=8.42,P<0.05),the proportion of cell necrosis increased(F=3.89,P<0.05),the content of macrophageassociated cytokines other than IL-4 increased(F=6.32-15.50,P<0.05),and the proportion of M1 and M2 types of macrophage in the immune microenvironment of hepatocellular carcinoma tumors was elevated(F=5.46,5.14,P<0.05).The gene expression and protein expression levels of cGAS-STING signaling pathway were elevated in hepatocellular carcinoma cells(mRNA expression of cGAS and STING:F=6.35,16.10,P<0.05;protein expression of cGAS and STING:F=71.31,37.15,P<0.05).Conclusions X-ray irradiation activates the cGAS-STING signaling pathway in hepatocellular carcinoma cells and contributes to the remodeling of the tumor immune microenvironment.