5-Aza-CdR联合EBNA1-DC疫苗诱导的淋巴细胞对鼻咽癌C666-1细胞的杀伤作用

Killing effects of 5-Aza-CdR combined with EBNA1-DC vaccine-induced lymphocytes on nasopharyngeal carcinoma C666-1 cells

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=0.0001)。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 to construct 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=0.0001).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.