弱酸洗脱肽负载的树突状细胞激活T细胞的实验研究

Activation of T lymphocytes in vitro by dendritic cells loaded with mild acid-eluted peptides

目的采用弱酸洗脱提取人卵巢癌细胞膜表面的肿瘤抗原肽,用于负载脐血树突状细胞(DCs),观察其对混合T淋巴细胞的体外激活效应。方法以淋巴细胞分离液分离脐血中的单个核细胞(CBMNC),将其中贴壁的细胞在细胞因子重组人粒巨噬细胞集落刺激因子(rhGM-CSF)、重组白介素(rhIL)-4及重组肿瘤坏死因子(rhTNF-α)的配伍作用下诱导分化为DCs。室温下以pH为3．3的枸橼酸-磷酸盐缓冲液处理人卵巢癌细胞,洗脱液经Oasis HLB小柱提取纯化后负载DCs。以3H-TdR掺入法测定负载抗原的DCs对混合T淋巴细胞的体外活化效应。结果贴壁的CBMNC在细胞因子2 000 U／mL rhGM-CSF、500 U／mL rhIL-4及100 U／mL rhTNF-α的配伍作用下体外诱导分化为具有典型形态和表型的DCs。人卵巢癌细胞弱酸洗脱肽负载的DCs与混合T淋巴细胞体外共培养后,T细胞增殖加快。结论CBMNC可以在GM-CSF、IL-4和TNF-α的作用下体外诱导分化为功能性DCs。pH为3.3的枸橼酸-磷酸盐缓冲液洗脱法可得到卵巢癌细胞的弱酸洗脱肽,用其负载脐血DCs,体外可激活细胞毒T淋巴细胞。

Objective To observe the in vitro activation effect of cord blood-derived dendritic cells（DCs） loaded with mild acid-eluted peptides of HO-8910 tumor cells on T lymphocyte. Methods Cord blood was collected from healthy term-idelivery mother. The cord blood mononuclear cells（CBMNCs） were separated by density gradient centrifugation（Ficoll-Hypaque）. The adherent fraction of CBMNCs was cultured in presence of relevant cytokines（rhGM-CSF 2 000 U/mL, rhIL-4 500 U/mL and rhTNF-α 100 U/mL） to generate DCs. The tumor cells of human ovarian carcinoma cell line HO-8910 were treated with citrate-phosphate buffer（pH 3.3） and the washing fluid was subjected to an Oasis HLB solid-phase extraction cartridge for mild acid-eluted peptides. As tumor antigens, mild-acid eluted peptides were used to prime the DCs in vitro. The stimulation ability of DCs on the T lymphocyte proliferation was reflected by mixed T lymphocyte reaction using ^3H-TdR incorporation. Results DCs with typical morphology and phenotype were harvested from the adherent fraction of CBMNCs in presence of rhGM-CSF 2 000 U/mL, rhIL-4 500 U/mL and rhTNF-α 100 U/mL in vitro. Loaded with the mild acid-eluted peptides, DCs were able to stimulate the amplification of allogeneic mixed T lymphocytes. The stimulate index（SI） of the loaded groups were higher than the unloading group at an effector ： target ratios of 1 ： 10, 1 ： 50 and 1 ： 100. Conclusion Functional DCs with typical morphology and phenotype had been successfully harvested from the adherent fraction of CBMNCs in presence of rhGM-CSF, rhIL-4 and rhTNF-α in vitro. After loading with mild acideluted peptides of ovarian carcinoma cells, the cord blood-derived DCs can activate CTL in vitro. （Shanghai Med J, 2007, 30：358-361）