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)、CD_(83)、CD_(86)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 CD_(83)、CD_(86)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。
Objective To investigate the effect of dendritic cells （DCs） pulsed by oliganucleotide containing ＂un-methylated cytimidine-phosphodiester bond-gnanylic 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 CD1α, CD63, CD86, and human leucocyte antigen-DR （HLA-DR） on the cell surface. ELISA was used to detect the IL-12 level in the supematant. T cells were obtained from the DCs. The suspensions of different groups of pulsed DCs were coincubated 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 CD1α among 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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第11期778-782,共5页
National Medical Journal of China
