黑素瘤患者树突状细胞体外快速培养成熟和临床应用研究

In vitro rapid generation and application of mature dendritic cells from peripheral blood cells of patient with melanoma

目的:建立从黑素瘤患者外周血体外快速稳定地诱导培养成熟树突状细胞(DC)的方法。方法:用密度梯度离心法分离患者外周血中分离出的单个核细胞(PBMC),再用贴壁法获取单个核细胞后加入粒-巨噬细胞集落刺激因子(GM-CSF)和白介素(IL)-4,随后分4组:第1和第2组分别在24h时加入多聚次黄嘌呤胞嘧啶核苷酸[Poly(I:C)]、肿瘤坏死因子(TNF)-α,继续培养24h,在48h时收集DC;第3和第4组分别在36h时加入Poly(I:C)、TNF-α,继续培养36h,在72h时收获DC。显微镜下观察DC形态,流式细胞术检测DC表型,体外同种混合淋巴细胞反应检测DC刺激T细胞的增殖活性。结果:经GM-CSF、IL-4、Poly(I:C)诱导培养72h获得大量成熟DC,形态学观察可见典型特征,荧光激活细胞分离器(FACS)检测表明,第3组DC高表达CD83、CD80、CD86和CD40;混合淋巴细胞反应(MLR)表明,第3组获得的DC具有较强的刺激同种淋巴细胞增殖能力。获取的成熟DC注射于患者浅表淋巴结后未出现明显不良反应。结论:Poly(I:C)体外诱导人外周血单个核细胞来源DC成熟的方法具有快速、稳定、经济、安全的特点,培养后DC回输患者体内未出现明显不良反应。

Objective： To get a rapid and stable method of generating mature Dendritic Cells from peripheral blood cells of patients with melanoma in vitro. Methods： Monocytes were isolated from normal human peripheral blood mononuclear cells （PBMCs） and were subgrouped into four groups and cultured with GM-CSF and IL-4. After 24h polyriboinosinic polyribocytidylic acid （Poly（I：C）） was added into the first group and TNF-α into the second group. DCs were collected at 48h. At 36h Poly（I：C） and TNF-α were added into the third and the last group, respectively. DCs were collected at 72h. The morphology was observed by microscope. The surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting （FACS）. T cell proliferating activity was determined by allogeneic MLR （mixed lymphocyte reaction） in vitro. Results： Large amounts of mature DCs could be obtained 72h after the cells in the presence of GM-CSF, IL-4 and Poly （I：C）.The induced DCs expressed high level of CD83, CD80, CD86 and CD40, and were potent to stimulate the proliferation of allogeneic lymphocytes. No significant side effect was found in these patients who received the injection of the mature DCs. Conclusion： The method of generating the mature DCs from PBMC in vitro induced by Poly （I： C） is rapid, stable, economical and safe.