肿瘤抗原致敏树突状细胞瘤苗治疗颅内胶质瘤的实验研究

Experimental study of bone-marrow-derived dendritic cells pulsed with tumor antigens in the treatment for intracranial gliomas

目的 研究肿瘤抗原致敏树突状细胞瘤苗治疗胶质瘤的疗效。方法 从大鼠的骨髓中加入GM CSF和IL 4培养树突状细胞 ,采用相差显微镜、扫描电镜观察了树突状细胞的形态学特点 ;免疫组织化学双标技术、流式细胞免疫学技术观察了其特异性抗体OX6和OX62 的表达 ;微酸洗脱方法抽提C6肿瘤抗原体外致敏树突状细胞 ,建立C6胶质瘤脑内动物模型 ,体内应用抗原致敏的树突状细胞 ,观察脑内肿瘤的疗效 ,并取致敏的动物的脾脏T细胞体外加入C6肿瘤抗原 ,加入IL 2培养进行CTL活性检测。结果 培养第 8天可见有典型的树突状细胞 ;免疫组织化学双标可见有OX6和OX62 阳性细胞 ,流式细胞免疫学分析发现OX62 阳性率为 93 0 3% ,OX6的阳性率为 92 0 1% ;体外细胞毒试验发现对相应的C6肿瘤细胞有明显的杀伤作用 ;动物实验发现实验组可见肿瘤组织内有大量的坏死 ,而对照组仅有少量的坏死 ,两者相比P <0 0 1。结论 体内注射采用微酸洗脱的抗原肽致敏树突状细胞瘤苗能够引起荷瘤动物脑肿瘤大面积的坏死 ,该方法为将来临床免疫治疗胶质瘤充分调动机体的免疫系统奠定基础。

Objective To investigate dendritic cells pulsed with tumor antigens in the treatment of intracranial gliomas when injected as a vaccine into tumor bearing animals. Methods Dendritic cells were isolated from rat bone marrow precursors stimulated in vitro with granulocyte macrophage colony stimulating factor (GM CSF) and interleukin 4 (IL 4). Cultured cell populations were confirmed by morphology with phase microscope and scanning electron microscope. Expressions of specific antibody (OX 6 and OX 62 ) were analyzed by double stain immunocytochemisty and flow cytometry. These dendritic cells were then pulsed (Coculture) ex vivo with acid eluted tumor antigens from C 6 glioma cells. We then established an intracranial glioma animal model treated with injections of either control media or C 6 tumor antigen pulsed dendritic cells. The rat brains were then removed and examined histologically, and spleens were harvested for cell mediated cytotoxicity assays.Results At 8 days, mature dendritic cells with typical cloved leaf shaped nucleus and long cytoplasmic dendrites, double stain immunocytochemisty showed expressions of OX 6 and OX 62 were positive, flow cytometric analysis of dendritic cells showed that 93 03% of cells were positive for OX 62 , whereas 92 01% for OX 6 (MHCⅡsurface marker). Cell mediated cytotoxicity assays showed that there were statistically significant compared with control cells. At necropsy, there were many more necrotic cells in tumor than control group (P<0 01). Conclusions Based on these results, dendritc cells pulsed with acid eluted peptides derived from autologous tumors could promote tumor necrosis. This represents a promising approach to the immunotherapy of established intracranial gliomas, which may serve as a basis for designing clinical trials in the patient with brain tumors.