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目的检测尤文肉瘤细胞A673和树突细胞(DC)融合构建的肿瘤疫苗对尤文肉瘤细胞株A673的杀伤作用。方法应用促融合剂PEG对尤文肉瘤A673细胞和从外周血单个核细胞(PBMC)诱生的树突细胞进行融合。利用细胞因子hGMCSF和hIL4从PBMC诱生DC,并对其表型进行流式细胞仪(FCM)分析,红色荧光染料PKH26标记A673细胞,绿色荧光染料PKH67标记DC,应用促融合剂PEG融合后光镜及电镜观察DC细胞和融合细胞形态,FCM检测融合效率,通过同种混合淋巴细胞反应检测其免疫刺激活性。通过IFNγELISA法产生细胞毒性T淋巴细胞(CTL)的量,通过51Cr细胞杀伤试验检测融合细胞对A673细胞的杀伤作用。结果FCM检测出从PBMC成功诱生出CD83、CD80、CD86及HLADR高表达的成熟DC。DCs/A673融合细胞的融合效率达到23%,同种混合淋巴细胞反应显示DCs/A673融合细胞有很强的免疫刺激活性。IFNγ分泌检测显示DCs/A673融合细胞组较对照组产生CTL水平显著增高。51Cr释放法检测融合细胞体外诱导抗原特异的CTL,融合细胞激活的CTL对肿瘤细胞系A673细胞的杀伤作用强于DCA673混合组、DC组、A673组的CTL(P<0.05),作用较对照各组显著增强。结论DC与A673细胞融合体外致敏自体T淋巴细胞能生成抗原特异CTL对尤文肉瘤细胞A673有一定的杀伤作用。
Objective To investigate the killing effect of tumor vaccine constructed by fusion of Ewing’s sarcoma A673 and dendritic cells (DCs) on Ewing’s sarcoma cell line A673. Methods Fusion dendritic cells induced by eucaryotic sarcoma A673 cells and peripheral blood mononuclear cells (PBMCs) were fused with PEG. DCs were induced from PBMC with cytokines hGMCSF and hIL4, and their phenotypes were analyzed by flow cytometry (FCM). The red fluorescent dye PKH26 labeled A673 cells, the green fluorescent dye PKH67 labeled DC, and the fusion fused with PEG The morphological changes of DC cells and fusion cells were observed under microscope and electron microscope. The fusion efficiency was detected by FCM and the immunostimulatory activity was detected by allogeneic mixed lymphocyte reaction. The amount of cytotoxic T lymphocytes (CTL) produced by the IFNγ ELISA method was used to detect the cytotoxicity of the fused cells to A673 cells by 51Cr cell killing assay. Results FCM showed that mature DCs with high expression of CD83, CD80, CD86 and HLADR were successfully induced from PBMCs. The fusion efficiency of DCs / A673 fusion cells reached 23%, allo-mixed lymphocyte reaction showed that DCs / A673 fusion cells had strong immunostimulatory activity. IFNγ secretion assay showed that CTL levels in DCs / A673 fusion cell group were significantly higher than those in control group. 51Cr release assay was used to detect the CTL induced by fusion cells in vitro. CTLs activated by fusion cells were more potent than DCA673 mixed group, DC group and A673 group (P <0.05) Group significantly increased. Conclusions The fusion of DCs with A673 cells in vitro induces autologous T lymphocytes to produce antigen-specific CTLs, which have a certain killing effect on Ewing’s sarcoma cells A673.