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目的:制备可控性大鼠卵巢癌树突状细胞(DCs)活疫苗并检测其免疫生物活性。方法:从大鼠Fischer344骨髓前体细胞中分离、扩增DCs,与转导有自杀基因Ⅰ型单纯疱疹病毒胸腺嘧啶核苷激酶(HSV1-tk)基因的大鼠卵巢癌细胞株NuTu-19/tk融合制成活疫苗。以流式细胞仪、激光共聚焦显微镜分析此活疫苗的生物学特征;LDH释放法检测其细胞毒活性(CTL);MTT法测定其对丙氧鸟苷(GCV)的反应性。结果:细胞融合率为28.14%,融合细胞呈典型的双阳性表现,高表达表面分子MHC-Ⅱ(OX6)、B7-2、OX62和ICAM-1。CTL结果显示活疫苗组对亲本肿瘤细胞的杀伤活性最强,死疫苗组次之,DCs与NuTu-19/tk混合组最差(P<0.01)。100μg/mlGCV对活疫苗的杀伤率为88.42%。结论:卵巢癌DC融合活疫苗在体外可产生明显的抗肿瘤免疫效应,并可通过自杀基因的杀伤作用将其在体外灭活。将自杀基因系统引入肿瘤活疫苗,为制备有效而安全的肿瘤疫苗提供了新思路。
Objective: To prepare controllable ovarian cancer dendritic cells (DCs) live vaccine and test its immunobiological activity. Methods: DCs were isolated from rat Fischer344 bone marrow progenitor cells and transfected into rat ovarian cancer cell line NuTu-19 / HSC1 transfected with suicide gene HSV1-tk, tk fusion made of live vaccine. The biological characteristics of the live vaccine were analyzed by flow cytometry and confocal laser scanning microscopy. The cytotoxicity (CTL) was measured by LDH release assay. The reactivity to gonadoside (GCV) was determined by MTT assay. Results: The fusion rate was 28.14%. The fusion cells showed a typical double positive expression of surface molecules MHC-Ⅱ (OX6), B7-2, OX62 and ICAM-1. CTL results showed that the live vaccine group had the strongest cytotoxic activity on the parental tumor cells, followed by the dead vaccine group, and the DCs and NuTu-19 / tk mixed group were the worst (P <0.01). The killing rate of 100μg / ml GCV on live vaccine was 88.42%. Conclusion: The DC fusion live vaccine of ovarian cancer can produce obvious antitumor immunity effect in vitro, and it can inactivate in vitro by the killing effect of suicide gene. The introduction of suicide gene system into live tumor vaccine provides a new idea for the preparation of an effective and safe tumor vaccine.