【摘要】 目的探讨含有未甲基化"胞嘧啶-磷酸二酯键-鸟嘌呤（CpG）基序"的寡脱氧核糖核苷酸（CpG ODN）致敏人外周血来源树突状细胞（DC）在卵巢癌免疫治疗中的作用。方法应用 CpGODN2006联合肿瘤相关抗原 CA125体外冲击致敏 DCs,流式细胞技术检测 DC 膜表面 CD_1α、CD₈₃、CD₈₆和人白细胞 DR 抗原（HLA-DR）的表达,用 ELISA 测定 DC 培养上清液中白细胞介素（IL）-12的分泌水平;四唑盐（MTT）比色试验法检测活化 DCs 对细胞毒性 T 淋巴细胞（CTL）杀伤 OVCAR-3卵巢癌细胞株作用的影响。结果 CpG ODN2006联合 CA125在体外可明显激活人外周血来源 DCs,DC表面分子 CD₈₃、CD₈₆和 HLA-DR 表达的阳性细胞百分率[（85±4）%、（87±12）%和（92士7）%]明显高于未致敏组[（19±4）%、（67±9）%和（63±6）%]（P<0.01）;联合致敏后 DCs 培养上清液中白细胞介素（IL）-12分泌水平为（467±84）ng/L,与未致敏组[（60±9）ng/L]和单纯 CA125致敏组[（97±16）ng/L]相比差异有统计学意义（P<0.01）;联合刺激后的 DCs 可以促进 T 淋巴细胞的增殖,增殖活性显著高于未致敏组和单纯 CA125致敏组（P<0.01）,且相同效靶比下所诱导的 CTLs 对OVCAR-3卵巢癌细胞的杀伤活性显著强于未致敏组、单纯 CA125致敏组和单纯 CpG ODN 致敏组（P<0.01）。结论 CpG ODN 联合 CA125体外致敏 DC 可诱导产生对 OVCAR-3卵巢癌细胞的免疫杀伤作用,是一种临床应用前景良好的肿瘤免疫治疗方法。更多还原

【Abstract】 Objective To investigate the effect of dendritic cells（DCs）pulsed by oligonucleotide containing"un-methylated cytimidine-phosphodiester bond-guanylic acid"motif（CpG ODN）on ovarian carcinoma cells.Methods Dendritic cells were isolated form the peripheral monocytes and co-incubated with synthesized CpG2006,CAI125,important epithelial ovarian cancer-associated antigen,or CpG ODN+ CA125 for 72 h.Flow cytometry（FC）was used to detect the expression of CD_1α,CD₆₃,CD₈₆,and human leucocyte antigen-DR（HLA-DR）on the cell surface.ELISA was used to detect the IL-12 level in the supernatant.T cells were obtained from the DCs.The suspensions of different groups of pulsed DCs were co- incubated with T cells.Mixed lymphocyte reaction（MLR）was used to detect the proliferating activity of the T cells.Ovarian carcinoma cells of the line OVCAR-3 were added into the culture fluid of T cells with different effector-target ratios,and then unpulsed DCs,CpG ODN-pulsed DCs,CpG ODN+CA125-pulseed DCs,and CA125-pulsed DCs were added respectively.MTT colorimetry was performed to measure the A values of different wells so as to calculate the killing rate.Results（1）The expression levels of CD₆₃, CD₈₆,and HLA-DR on the membranes of the CpG ODN-pulsed DCs and CpG ODN+C125-pulsed DCs were significantly higher than those of the un-pulsed DCs（all P<0.01）,however,there were no significant differences in the expression of CD_1αamong different groups（all P>0.05）.（2）The IL-12 levels in the supernatants of the CpG ODN-pulsed DCs and CpG ODN+C125-pulsed DCs were significantly higher than those of the unpulsed and CA125-pulsed groups（all P<0.01）.（3）MLR showed that the T cell proliferation rates of the T cells sensitized by the CpG ODN-pulsed DCs and CpG ODN+C125-pulsed DCs were both higher than those of the T cells stimulated by the unpulsed and CA125-pulsed groups when the effector:target ratio was 10:1（all P<0.01）.（4）The killing rates on OVCAR-3 cells of the CTLs sensitized by CpG ODN+CA125-pulseed DCs at the same effector:target ratios were all higher than those of the CTLs sensitized by the CpG ODN-pulsed DCs,CA125-pulsed DCs,and unpulsed DCs（all P<0.01）, the killing activity of the cytotoxic T lymphocytes（CTLs）sensitized by CpG ODN + CA125-pulseed DCs on the OVCAR-3 cells was shown even when the effector:target ratio was as low as 10:1,and then increased along with the increase of the effector:target ratio to the height of 64.9%.Conclusion Capable of inducing immune cytotoxicity on ovarian carcinoma,DCs sensitized by CpG ODN+CA125 may have a great implication on clinical application.更多还原