Objective: To investigate the effect and associated mechanism of tumor tissue-infiltrating NK cells after receiving radiotherapy for hepatocellular carcinoma (HCC). Methods: A HCC tumor-bearing mouse model was constructed using human hepatocellular carcinoma cell line (SK-Hep-1) and divided into four groups: control, radiotherapy, NK cell clearance, and NK clearance combined with radiotherapy. Tumor growth condition was simultaneously recorded. The NK cell ratio in peripheral blood and the NK cell intratumoral infiltration condition were detected by flow cytometry and immunohistochemistry. Lentiviral-constructed SK-Hep-1 cells was used to detect the effect of radiotherapy on the regulation of CXCL10 and NK cell chemotaxis following EZH2 overexpression. SK-Hep-1 cells were irradiated in vitro and in vivo. The expression levels of EZH2 and CXCL10 mRNA and protein in the two groups of cell lines and mouse tumor tissues were detected by reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting (WB), and immunohistochemistry. The chemotaxis and blocking experiments were used to validate the chemotaxis effect of CXCL10 on NK cells. The independent sample t-test was used to compare the groups. P<0.05 was considered statistically significant. Results: The HCC tumor-bearing mouse model experiment showed that HCC tumor growth was most remarkable in the NK clearance combined with the radiotherapy group compared to the radiotherapy group (P<0.05). Compared with the control group, the number of NK cells in the peripheral blood of nude mice in the radiotherapy group was significantly reduced, while the NK cell intratumoral infiltration was significantly increased (P<0.05). Flow cytometry and immunohistochemistry showed in vitro and in vivo expressional alterations. The average expression levels of EZH2 mRNA and protein in hepatocellular carcinoma cell lines and tumor tissues were decreased in the radiotherapy group than the control group and mouse tumor tissues (P<0.05), while the mRNA and protein expression levels of CXCL10 increased (P<0.05). The cell supernatant following radiotherapy enhanced NK cell chemotaxis but inhibited CXCL10 neutralization. EZH2 overexpression validated that radiotherapy up-regulated CXCL10 mRNA and down-regulated protein expression levels in in vitro and in vivo experiments (P<0.05). The chemotactic effect on NK cells was significantly weakened with EZH2 overexpression following radiotherapy. Conclusion: NK cells, as immune effector cells, are directly involved in radiotherapy- activated anti-HCC immunity. Importantly, radiotherapy inhibits EZH2 expression in hepatocellular carcinoma, thereby upregulating CXCL10 expression and enhancing intratumoral NK cell invasion.
目的： 探讨肝细胞癌（HCC）在接受放射治疗后自然杀伤（NK）细胞在肿瘤组织中的浸润作用及其相关机制。 方法： 应用人肝癌细胞系SK-Hep-1构建HCC荷瘤小鼠模型，分为4组：对照组、放射治疗组、NK细胞清除组及NK清除联合放射治疗组，同时记录肿瘤生长情况。应用流式细胞术及免疫组织化学检测小鼠外周血NK细胞比率及肿瘤组织内NK细胞浸润情况。应用慢病毒构建Zeste同源增强子2（EZH2）过表达的SK-Hep-1细胞，并检测放射治疗后EZH2过表达细胞的CXC趋化因子配体10（CXCL10）的表达量及对NK细胞的趋化作用。应用逆转录聚合酶链式反应（RT-PCR）、酶联免疫吸附试验（ELISA）、蛋白免疫印迹法（WB）及免疫组织化学检测评价SK-Hep-1细胞在体外、体内进行照射处理后EZH2、CXCL10 mRNA和蛋白的表达水平，通过趋化及阻断实验验证CXCL10对NK细胞的趋化作用。组间的比较采用独立样本t检验。P<0.05表示差异具有统计学意义。 结果： HCC荷瘤小鼠模型实验提示，与放射治疗组相比，NK清除联合放射治疗组小鼠HCC肿瘤生长差异更显著（P<0.05）；与对照组相比，放射治疗组小鼠外周血NK细胞数显著减少，而瘤内NK细胞浸润显著增加（P<0.05）。流式细胞术及免疫组织化学检测体内体外的表达量实验提示，与对照组SK-Hep-1细胞及小鼠肿瘤组织相比，放射治疗组肝癌细胞系及肿瘤组织EZH2 mRNA和蛋白表达水平均下降（P值均<0.05），CXCL10 mRNA和蛋白表达水平均升高（P值均<0.05）。放射治疗后的细胞上清液对NK细胞趋化作用增强，中和CXCL10后抑制了NK细胞趋化作用。EZH2过表达体内外实验证实，放射治疗可通过下调EZH2表达上调CXCL10 mRNA及蛋白表达水平（P值均<0.05）。放射治疗处理后，EZH2过表达对NK细胞趋化作用明显减弱。 结论： NK细胞作为免疫效应细胞直接参与放射治疗激活的抗HCC免疫过程。放射治疗通过抑制HCC中EZH2表达，上调CXCL10表达量从而增强NK细胞瘤内浸润。.