目的：探讨EB病毒核抗原1（EBNA1） mRNA修饰的DC（EBNA1-DC）诱导的淋巴细胞联合甲基化抑制剂5-Aza-CdR对鼻咽癌C666-1细胞的杀伤作用。方法：以构建的EBNA1-pCDNA3.1质粒为模板，体外转录获得EBNA1 mRNA，通过脂质体转染至健康人外周血来源DC，构建EBNA1-DC疫苗。流式细胞术检测转染后DC表型及5-Aza-CdR 处理后的C666-1 细胞凋亡情况。实时无标记动态细胞分析技术检测EBNA1-DC 疫苗诱导的淋巴细胞联合5-Aza-CdR 的特异性抗肿瘤活性。结果：转染EBNA1 mRNA 后EBNA1-DC 表面EBNA1 阳性率为（59.3±5.85）%，HLA-DR 的表达与未转染DC 相比显著升高[（84.9±5.5）% vs（68.0±5.8）%，P=0.026]，CD80 的表达也显著升高[（88.2±3.9）% vs （61.1±4.4）%，P=0.015]。低剂量5-Aza-CdR 处理后的C666-1细胞凋亡情况与未处理的细胞相比无显著差异。经低浓度5-Aza-CdR 预处理的C666-1细胞中IRF7基因表达与未处理的细胞相比显著升高（P=0.000 1）。与空载的DC相比，EBNA1-DC诱导的淋巴细胞对EBV 阳性表达的C666-1细胞具有更强的特异性杀伤活性（P=0.049）；经低浓度5-Aza-CdR 预处理的C666-1 细胞对EBNA1-DC 诱导的特异性免疫杀伤更敏感（P=0.019）。结论：5-Aza-CdR 与EBNA1-DC疫苗联合可显著增强对C666-1细胞的特异性免疫杀伤，本研究为开拓以mRNA为基础的DC疫苗及其在临床综合治疗中的应用转化提供前期研究基础。

Objective: To investigate the killing effects of Epstein-Barr virus nuclear antigen 1 (EBNA1) mRNA-modified DC (EBNA1-DC) -induced lymphocytes combined with methylation inhibitor 5-Aza-CdR on nasopharyngeal carcinoma C666-1 cells.Methods: EBNA1-pCDNA3.1 plasmid was used as a template, and EBNA1 mRNA was obtained by in vitro transcription.Subsequently, EBNA1 mRNA was transfected into dendritic cells derived from peripheral blood from healthy donors by liposome toconstruct EBNA1-DC vaccine. Flow cytometry was used to detect the transfected DC phenotype and the apoptosis of C666-1 cells after 5-Aza-CdR treatment. Real-time cell analysis was used to detect the specific antitumor activity of EBNA1-DC vaccine-induced lymphocytes combined with 5-Aza-CdR. Results: The positive rate of EBNA1on the surface of EBNA1-DC after transfection with EBNA1 mRNA was (59.3±5.85) %. The expression of HLA-DR was significantly higher than that of untransfected DC ([84.9±5.5]% vs [68.0±5.8]% , P=0.026). The expression of CD80 was significantly improved from (88.2±3.9)% to (61.1±4.4)% (P=0.015). The apoptosis of C666-1 cells treated with low-dose 5-Aza-CdR was not significantly different from that of untreated cells. The expression of IRF7 gene in C666-1 cells pretreated with low-dos 5-Aza-CdR was significantly higher than that in untreated cells (P=0.000 1).Lymphocytes induced by EBNA1-DC had stronger specific killing activity against EBV+C666-1 cells compared with untransfected DC (P=0.049). C666-1 cells pretreated with low dose 5-Aza-CdR were more sensitive to specific immune killing induced by EBNA1-DC (P=0.019). Conclusion: The combination of 5-Aza-CdR and EBNA1-DC vaccine can significantly enhance the specific immune killing effect on C666-1 cells. This study provides the preliminary research basis for the development of the mRNA-DC vaccine and its application in clinical comprehensive therapy.

福建省科技厅自然基金项目（No. 2022J011055）；福建省卫生健康委员会青年基金项目（No. 2020QNB008）；福建省肿瘤医院院内课题（No. 2021YN07）；福建省肿瘤医院院内课题（No. 2021YN11）；厦门市医疗卫生指导性项目（No. 3502Z202009075）