脐血来源树突状细胞的体外扩增、鉴定及冻存

Proliferation, identification and cryopreservation of dendritic cells from umbilical cord blood in vitro

目的研究脐血来源树突状细胞（DCs）的体外扩增、鉴定和冻存。方法用Ficoll分离获得脐血单个核细胞，以重组hGM—CSF、hIL-4及hTNF-α体外诱导培养14d。在DCs发育过程中，在光镜和电镜下观察其生长情况，应用流式细胞仪检测DCs表型。采用台盼蓝拒染法测定DCs增殖水平，MTT法检测DCs活化的T细胞对肿瘤细胞的杀伤性。同时用二甲亚砜作为保护剂，DCs经过-20℃和-80℃降温，在-196℃液氮保存，然后40℃水浴复苏。结果第6天体外培养的DCs由贴壁状态变为悬浮毛刺状细胞，随着培养时间的延长，此类细胞数量增多，第14天为形态不规则的毛刺状，为典型树突状细胞形态。流式细胞仪检测表明，成熟DCs高水平地表达HLA—DR、CD80、CD83、CD1a、CD11c。被激活的T细胞对2种来源于不同组织的肿瘤细胞均产生了明显的杀伤性。冻存DCs和新鲜DCs的生物活性无明显差异。结论脐血单个核细胞细胞经hGM—CSF＋hIL-4＋hTNF—α体外培养，能诱导出DCs，为进一步开展DCs的实验研究与临床应用奠定了基础。

Objective： To investigate the proliferation, identification and cryopreservation of dendritic cells derived from human cord blood progenitor cells in vitro. Methods： The mononuclear cells were prepared from cord blood with Ficoll centrifugation method, and then induced with the recombinant cytokines hGM-CSF, hIL-4 and hTNF-α for 14 days. We observed the DCs growth and determined DCs phenotypes by flow cytometry. The proliferation of DCs were determined by trypan blue exclusion assay and the capacity of initiating T cell-depent anti-tumor immune responses by MTT. DMSO was added into culture medium as cryopreservation reagent. After freezing in -20℃ and -80℃ refrigerator, dendritic cells were finally cryopreserved in -196℃ liquid nitrogen, and then thawed with 40℃ water. Results, The dendritic cells cultured in vitro turned into suspensive growth from adhesive situation at d6, along with the incubation, the number of DCs increased and the cells showed the irregular morphologic appearance of DCs with veiled edges in the 14th day. It was showed that the mature DCs could express the relatively specific marker such as HLA-DR, CD80, CD83, CD1a and CD11c by FACS. T cells activated by DCs showed magnificent cytotoxicity on 2 different kinds of tumor cells cOmpared with respective control groups. There was no obvious difference in biological activity between cryopreserved and fresh DCs. Conclusion： The dendritic cells can be induced from human cord blood that is co-incubated with hGM-CSF, hIL-4 and hTNF-α in vitro. The investigation establishes a foundation for further research and clinical use of DCs.