冻融人外周血树突状细胞基因疫苗体外抗肿瘤活性研究

目的观察EB病毒潜伏膜蛋白重组腺病毒(rAd-EBV-LMP2)转染的冻融人外周血树突状细胞(DC)诱导的特异性细胞毒性T淋巴细胞(CTL)抗肿瘤活性。方法人外周血来源的单核细胞经细胞因子扩增培养为成熟DC,液氮冻存;rAd-LMP2重组腺病毒转染冻融DC制备疫苗。动态观察细胞形态学特征,四甲基偶氮唑蓝(MTT)法检测DC疫苗刺激同种T淋巴细胞增殖活性,以及诱导的CTL对肿瘤细胞的杀伤活性。结果rAd-LMP2转染的冻融DC疫苗具有形态迥异、多突起的典型形态学特征,其刺激同种T淋巴细胞的增殖能力明显高于对照组(P<0.01),能诱导高效的CTL活性(P<0.01),与新鲜DC疫苗差异无统计学意义(P>0.05)。结论rAd-LMP2转染冻融DC疫苗在体外能激发较强的EBV-LMP2特异性的功能性CTL,为EBV相关的鼻咽癌(NPC)的免疫治疗提供策略。

Antitumor activity induced by human cryopreserved dendritic cell transfected with viral antigen gene

Objective To observe the antitumor effects of EBV-LMP2 specific CTL induced by human cryopreserved dendritic cells （DCs） transfected with recombinant adenovirus in vitro. Methods Mature DCs derived from human peripheral blood mononuclear cells （PBMC） were induced by cytokines, and then cryopreserved in liquid nitrogen. DC vaccine was prepared by transfecting frozen DCs with EBV- LMP2 recombinant adenovirus. The morphology of DC was observed dynamically. T lymphocytes proliferation stimulated by rAd-LMP2-DC vaccine and cytotoxicity of EBV- LMP2 specific CTL were determined with MTT assay respectively. Results Cryopreserved DC vaccine had typical morphologic characteristics with irregular dendritic projections. Stimulation index （SI） of T lymphocytes by DC vaccine was significantly higher than those of the control groups （P〈0.01）. Potent LMP2-specific antitumor CTL could be induced by cryopreserved DC vaccine （P〈0.01）. There was no significant difference between cryopreserved DC vaccine and fresh DC vaccine （P〉0.05）. Conclusion Cryopreserved DC vaccine transfected with adenovirus encoding LMP2 could elicit strong and functional EBV-LMP2 specific CTL in vitro. This study could provide a rational strategy for immunotherapy of EBV associated NPC.