外周血单核细胞来源树突状细胞体外扩增及诱导抗乳腺癌免疫的研究

目的研究外周血来源树突状细胞(DC)的体外扩增及诱导特异性抗乳腺癌细胞的免疫效应。方法(1)从外周血中分离单个核细胞后,获得单核细胞(monocyte,Mo)。粒-单集落刺激因子(GM-CSF)和白介素4(IL-4)诱导分化扩增培养7d后,应用流式细胞术进行表型分析。(2)诱导单核细胞分化的第3天加入人乳腺癌细胞株3A0的冻融抗原培养4d后,获得负载肿瘤抗原的成熟DC;将致敏DC与从外周血中分离的同种异体T淋巴细胞共培养3d,获得细胞毒T淋巴细胞(CTL);四甲基偶氮唑蓝法检测CTL及上清液对人乳腺癌细胞株3A0、人胚肾细胞株293T(对照细胞)、人肝癌细胞株HCCC-9810的细胞毒作用。结果(1)外周血来源Mo在GM-GSF和IL-4作用下,第7天后可分化生成成熟的DC,高表达DC特异性抗原CDla、CD80(B7-1)、CD86(B7-2)HLA-DR、CD83。(2)DC可负载并递呈肿瘤抗原,激活同种异体T淋巴细胞,诱导肿瘤特异性CTL产生。不同浓度CTL及上清液对乳腺癌细胞3A0有特异性杀伤、抑制作用(P<0.05)。结论外周血中Mo可体外分化扩增为成熟的功能性DC,并诱导出特异性杀伤乳腺癌细胞的免疫效应。

A study of generation of monocyte-derived dendritic cell from peripheral blood and inducement of immunity of bresat neoplasm

Objective To investigate the ability of generation of dendritic cell (DC) from precursor in peripheral blood and inducing specific antitumor immune response.Methods (1)The monocytes(Mo) were isolated from peripheral blood and cultured in medium with granulocyte-macrophage colony stimulating factor(GM-CSF) and interleukin 4(IL-4).Cells were harvested and detected for phenotypic analysis by FACS 7 days later.(2)The mature DC with specific tumor-associated antigen(TAA) was collected on day 7.After cultured for 3d,the mature DC could activate allogeneic T cells to become tumor specific cytotoxic T lymphocytes(CTL).Methyl thiazolyl tetrazolium(MTT) assay was employed to detect inhibition rate of CTL and its supernatant on 3A0 cells,HCCC-9810 cells,293T cells,respectively.Results (1)DC precursors in peripheral blood could be induced to differentiate and mature in medium containing GM-CSF/IL-4 for 7d.The induced DC had high expression of CDla,CD80(B7-1),CD83,CD86,(B7-2),and HLA-DR. (2)Mature DC with TAA could activate naive T cells to become tumor-specific CTL.Different cell amounts of CTL had significant inhibition or killing response on 3A0 cells(P<0.05).Conclusion The Mo from peripheral blood can be induced to be functional DC,which can activate naive T lymphocyte to CTL and induce specific antitumor immune response.