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目的 :探讨人外周血单核细胞来源的树突状细胞 (Dendriticcells ,DC)与人白血病细胞K562融合后 ,能否诱导出 p210蛋白特异性的CTL ,为DC疫苗的临床应用提供理论基础。方法 :外周血来源的贴附单核细胞 ,在人GM CSF(1000U/ml)和IL 4(1000U/ml)作用下 ,培养5～7天后 ,与人白血病细胞K562进行融合 ,融合细胞再与自体的淋巴细胞共同孵育10～14天 ,采用乳酸脱氢酶释放试验分析其对 p210蛋白阳性细胞的杀伤效果。为显示融合效果 ,对照试验组K562细胞用绿色荧光蛋白 (GFP)进行标记。结果 :贴附的单核细胞在GM CSF和IL 4作用下 ,培养5天后 ,约有70 %的DC产生 ,流式细胞仪分析表明 ,这些细胞为HLA DR、HLA A ,B ,C以及CD1α阳性 ,并高表达共刺激分子CD80和CD86。DC与GFP标记的K562细胞融合24h后 ,表达GFP蛋白的细胞中约有60 %呈现出典型的DC形态特征。乳酸脱氢酶释放试验结果表明 ,融合细胞能激活淋巴细胞产生 p210蛋白特异性的CTL。结论 :树突状细胞与肿瘤细胞融合后 ,能诱导出肿瘤特异性的CTL ,显示出DC疫苗抗肿瘤作用的广泛前景。
Objective : To investigate whether the fusion of human peripheral blood mononuclear cell-derived dendritic cells (DCs) with human leukemia cell K562 can induce p210 protein-specific CTLs, providing a theoretical basis for the clinical application of DC vaccines. Methods: Peripheral blood-derived adherent mononuclear cells were cultured for 5 to 7 days under the action of human GM CSF (1000 U/ml) and IL 4 (1000 U/ml), fused with human leukemia cell K562, and fused with cells. The autologous lymphocytes were co-incubated for 10 to 14 days and the killing effect on p210 protein-positive cells was analyzed using a lactate dehydrogenase release assay. To show the fusion effect, K562 cells in the control group were labeled with green fluorescent protein (GFP). RESULTS: After 5 days of culture with attached GM CSF and IL-4, approximately 70% of the DCs were produced in the attached mononuclear cells. Flow cytometric analysis showed that these cells were HLA DR, HLA A, B, C, and CD1α. Positive and highly expressed co-stimulatory molecules CD80 and CD86. After fusion of DC with GFP-tagged K562 cells for 24 h, approximately 60% of the cells expressing GFP protein exhibited typical DC morphology. Lactate dehydrogenase release assays have shown that fused cells can activate lymphocytes to produce p210 protein-specific CTLs. Conclusion : After fusion of dendritic cells with tumor cells, tumor-specific CTLs can be induced, showing the broad prospects of anti-tumor effects of DC vaccines.