人脐血树突状细胞分泌外来体的特性及其增强CTL杀伤活性的研究

The properties of exosomes secreted by human umbilical cord blood dendritic cells and its enhancement of CTL cytotoxicity

目的探讨脐血树突状细胞(UBDC)及其分泌的亚细胞结构外来体(exosomes)的特性,以及他们对细胞毒性T细胞(CTL)杀伤活性的增强作用。方法分离脐血单个核细胞(UBMC),用rhSCF、rhGM-CSF及rhIL-4诱导12d,加入经反复冻融提取的肿瘤细胞膜抗原,继续培养2d。收集DC并从其培养上清中通过离心(1×105g)分离外来体。用流式细胞术、SDS-PAGE、透射电子显微镜(TEM)和MTT比色法,对DC和外来体的特性及其对CTL杀伤活性的增强作用进行分析。结果用3种细胞因子体外诱导UBMC12d,可使其扩增10倍,并出现DC典型的形态特征。负载肿瘤抗原后,细胞表面可高表达MHC-I、MHC-II、CD40、CD80、CD86、CD11c和CD54。利用超速离心法能从DC培养上清中分离出外来体。混合淋巴细胞反应(MLR)和CTL杀伤活性的分析表明,脐血DC分泌的外来体能有效地刺激T细胞增殖并增强CTL的杀伤活性。结论由UBMC可诱导出大量的DC并分泌外来体。后者可有效地向CTL细胞递呈肿瘤抗原使之活化并增强其杀伤活性。

AIM： To investigate the properties of exosomes secreted by dendritic cells （DCs） derived from human umbilical cord blood mononuclear cells（HUBMCs） and its enhancement of CTL cytotoxicity. METHODS： The HUBMCs were separated from human umbilical cord blood （HUCB）. The generation of DCs were induced for 12 days in medium containing SCF, IL-4 and GM-CSF. Tumor antigen extracted by repeated freeze-thawing were added to culture medium and cultured successively for 2 days. The DCs were collected and exosomes were separated from culture supernatant through centrifugation at 100 000 g. The exosomes-associated properties were analyzed by FCM, SDS-PAGE, transmission electron microscope（TEM）and MTT colorimetry. RESULTS： After being induced for 12 days with SCF, IL-4 and GM-CSF the number of DCs had a 10-fold increase and displayed typically morphological characteristics of DCs. FCM analysis showed that the DCs loaded with tumor antigen expressed highly MHC-I, MHC-II, CD40, CD80, CD86, CD11c and CD54. Exosomes could be separated from culture supernatant of DCs by ultracentrifugation. Mixed lymphocyte reation（MLR） and cytotoxicity assay showed that exosomes secreted by DCs could effectively stimulate T cell proliferation and enhance their cytotoxicity. CONCLUSION： A large number of DCs are obtained from UBMCs and the exosomes are secreted from them in vitro. The exosomes can present tumor antigen to T cells and en- hance the cytotoxicity of CTLs.