【摘要】 目的探讨负载卵巢癌冻融抗原对树突状细胞分泌细胞因子的影响。方法采用反复冻融法获得卵巢癌细胞株SKOV3细胞抗原,联合应用重组人粒细胞-巨噬细胞集落刺激因子、重组人白细胞介素-4和重组人肿瘤坏死因子-α,体外诱导人外周血CD14+单核细胞为成熟DCs并负载卵巢癌肿瘤抗原;采用ELISA法检测培养细胞上清液中分泌IL-12p70、IFN-γ和IL-10含量,评价非成熟DCs和成熟DCs以及肿瘤抗原负载DCs和未负载肿瘤抗原DCs激活的CTL分泌细胞因子的能力。结果联合应用重组人的GM-CSF、IL-4和TNF-α可在体外诱导出成熟DCs;经ELISA方法检测,不同状态下的DCs分泌IL-12p70、IFN-γ的量不同,成熟DCs较非成熟DCs有更强的分泌IL-12p70、IFN-γ的能力(P<0.05),负载抗原DCs激活的CTL较未负载抗原DCs激活的CTL有更强的分泌IL-12p70[(182.89±4.57)pg/ml和(99.76±5.42)pg/ml,P<0.01]和IFN-γ的能力[(102.11±5.95)pg/ml和(68.29±5.04)pg/ml,P<0.01]。而成熟DCs分泌IL-10的能力与非成熟DCs相比,差异无统计学意义(P>0.05)。负载抗原后DCs激活的CTL与未负载抗原DCs激活CTL上清液中IL-10的浓度相比,两者之间的差异无统计学意义(P>0.05)。结论细胞因子的分泌量受DCs的成熟状态及某些刺激信号的影响,卵巢癌冻融抗原是其刺激信号之一。更多还原

【Abstract】 Objective To detect the effect of the SKOV3 ovarian carcinoma cells lysate on the cytokine-secreting capacity of the dendritic cells (DCs).Methods Soluble antigens were obtained from SKOV3 ovarian carcinoma cells lysate by cycling freezing-thawing. DCs were generated from human peripheral blood monocytes,which were isolated from peripheral blood of healthy donors by ficoll-hypaque gradient centrifugation and purified by using MACS CD14 isolation kit. To induce DCs,the purified CD14+ cells were cultured in the presence of granulocyte/macrophage colony-stimulating factor and interleukin(IL)-4/tumor necrosis factor-α in vitro for 12 days. The DCs were loaded with antigen generated from SKOV3 ovarian carcinoma cells. Concentrations of cytokine (IL-12p70,IFN-γ and IL-10) in the cell culture supernatants were quantified by enzyme-linked immunosorbent assay(ELISA).Results PBMCs from healthy volunteer could be induced into DCs in vitro. Supernatants of different condition DCs were analyzed for cytokine (IL-12p70,IFN-γ and IL-10) secretion. Interestingly,we found that IL-12p70 and IFN-γ secretion were up-regulated in the supernatant of CTL which was activated by SKOV3 ovarian carcinoma cells lysate stimulated DCs (182.89±4.57) pg/ml versus(99.76±5.42) pg/ml (P<0.01) and (102.11±5.95) pg/ml versus (68.29±5.04) pg/ml(P<0.01). DCs secreted significantly higher amount of IL-12 and IFN-γ than immature DCs (P<0.05),but the IL-10 secretion was not significantly different between the two groups.Conclusion The cytokine secreting capacity was affected by maturation status and extracellar stimulation signals of antigens from the SKOV3 ovarian carcinoma cells lysate as well.更多还原