含癌胚抗原片段的重组腺相关病毒转染树突状细胞疫苗的体外杀伤作用

The killing effect in vitro of dendritic cells vaccine transfected by recombinant adeno-associated virus with carcinoembryonic gene fragment

目的:观察研究人外周血单核细胞来源的树突状细胞(DC)转染含癌胚抗原(CEA)片段的重组腺相关病毒后所诱导的特异性T细胞对直肠癌细胞株LOVO和SW480的体外杀伤作用。方法:抽取HLA表型为A11的健康志愿者外周血,分离单核细胞,体外培养,使用含CEA片断的重组腺相关病毒转染未成熟DC,诱导特异性T细胞。检测体外培养的DC和CTL活性,并使用MTT法检测细胞毒性T细胞(CTL)对LOVO细胞的杀伤作用。结果:转染或未转染的体外培养的成熟DC高表达CD40、CD86、IL-12,诱导的细胞毒性T细胞高表达IFN-γ;转染后DC诱导特异性细胞毒性T细胞可有效识别并杀伤HLA-A11阳性的LOVO细胞。结论:重组腺相关病毒转染DC,不明显改变DC表型和刺激淋巴细胞增殖、分化功能,可诱导自体细胞毒性T细胞增殖,含CEA片断的腺相关病毒转染DC诱导自体细胞毒性T细胞对LOVO细胞有明显杀伤作用,DC疫苗可以作为直肠癌患者免疫治疗的有效补充。

Objective Dendritic cells vaccine is one of the important active immunotherapies for neoplasm. We observe the killing effect in vitro of cytotoxic T lymphocytes （CTL）, a specific T cell, on the colorectal carcinoma LOVO and SW480 cells. CTL is induced by human peripheral blood monocyte-originated dendritic cells （DC）which have been transfected by recombinant adeno-associated virus （rAAV） with carcinoembryonic （CEA） gene fragment. Methods Immature DCs were generated from peripheral blood monocytes in healthy volunteers with HLA-All positive, and then transfected by rAAV with CEA gene fragment. CTL was thereafter induced. The activity of DC and CTL was measured and the killing effect of CTL on LoVo cells was detected using MTT assay. Results The mature DC which were transfected or not highly expressed CD40, CD86 and IL-12. IFN-γ was highly expressed in the CTL. The DC-induced CTL could effectively recognize and destroy the LOVO cells with HLA-All positive, Conclusion DC transfected by rAAV can stimulate the proliferation and differentiation of lymphocyte and also induce the proliferation of CTL, and their own phenotypes are not altered. DC vaccine can effectively be used as an adjuvant immunotherapy for patients with colorectal carcinoma.