摘要
目的研究膀胱癌患者外周血树突状细胞(DC)的体外培养扩增和鉴定。方法应用Ficoll-Hypaque离心获得界面细胞,贴壁培养2h,获得单个核细胞,体外以重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF,50ng/ml)+重组人白细胞介素4(rhlL-4,10ng/ml)+人肿瘤坏死因子-α(hTNF-α,50ng/ml)诱导培养。倒置相差显微镜及扫描电镜下观察DC生长情况；流式细胞仪检测DC表型；MTT法检测DC活化的T细胞对肿瘤细胞BIU87的杀伤率。结果第6天体外培养的DC由贴壁状态变为悬浮毛刺状细胞,第8天为形态不规则的毛刺状,为典型DC形态。流式细胞仪检测表明,成熟DC高水平表达CD_(1a)、CD_(83)、CD_(86)及HLA-DR等。被DC激活的T细胞对膀胱癌细胞株BIU87的杀伤率为(48.8±3.7)%,未经DC激活的T细胞的杀伤率为(25.7±1.5)%,2组比较差异有统计学意义(P＜0.01)。结论膀胱癌患者外周血经rhGM-CSF+rhIL-4+hTNF-α体外诱导培养能诱导出DC。
Objective To investigate the proliferation and identification of dendritic cells （DC） derived from peripheral blood of patients with bladder cancer in vitro. Methods The mononuclear cells were prepared from peripheral blood of patients with bladder cancer by Ficoll-Hypaque centrifugation method, and were induced by the recombinant cytokines hGM-CSF （ 50 ng/ml） , hIL-4 （ 10 ng/ml） and hTNF-α （ 50 ng/ ml） for 2 weeks. The growth and morphology of DC were observed through the phase contrast or electron microscope, and their phenotypes were determined by flow cytometry. The capacity of DC to activate T cell-dependent anti-tumor immune responses was tested by MTT method. Results The DC cultured in vitro turned into suspensive growth from adhesive situation on the 6th day, then the number of DC increased continuously and the cells showed the irregular morphologic appearance of DC with veiled edges on the 8th day. Flow cytometry showed that the mature DC expressed high levels of specific markers such as CD1a,CD83, CD86 and HLA-DR. T cells activated by DC showed strong cytotoxicity to bladder cancer cell line BIU87 with a killing rate of （48.8 ± 3.7）% ,while the killing rate of T cells which were not activated by DC was （25.7 ± 1.5） % ;the difference of the rate between them was significant （P 〈 0.01 ）. Conclusions The DC can be cultured from peripheral blood of patients with bladder cancer by induction of rhGM-CSF,rhIL-4 and hTNF-α in vitro. This may lay an experimental foundation for further research on DC vaccine.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
2006年第7期472-474,共3页
Chinese Journal of Urology
