Tong Xiang-min, Jin Jie, Qian Wen-bin, Meng Hai-tao, Xue Yong-quan
The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2005 Jul;34(4):348-52, 357. doi: 10.3785/j.issn.1008-9292.2005.04.013.
To induce primary chronic myeloid leukemia (CML) cells into dendritic cells (DCs).
Bone marrow mononuclear cells (MNCs) were isolated from 13 CML patients and peripheral blood MNCs from 5 healthy donors. The isolated MNCs were co-cultured with rhGM-CSF 1,000 U/ml, rhIL- 4,500 U/ml and TNF-alpha 50 U/ml for 10 days. The morphological features were observed by Wright's staining,inverted microscope and electron microscope. CD(80), CD(86), CD(83), CD(1a) and HLA-DR expression were assayed by flow cytometry, cytogenetic analysis was performed by fluorescence in-situ hybridization(FISH). The concentration of IL-12 was measured by ELISA and the function of antigen presenting was tested by mixed lymphocyte reaction (MLR).
After being cultured with cytokines, the typical dendritic appearance with delicate membrane projections was observed. The CD(80), CD(86), CD(83), CD(1a) and HLA-DR markers and capacity of stimulating allogeneic T cells were upregulated significantly. FISH confirmed that the DCs were generated from leukemic origin and CML DCs could secrete higher level of IL-12 than CML MNCs. There were no differences in morphology and immunophenotype expression between DCs derived from CML and those from normal individuals. However, DCs from CML patients displayed weaker activity than that of normal individuals when tested in MLR.
CML cells could be induced into leukemia-DCs by co-culture with cytokines.
将原发性慢性髓性白血病(CML)细胞诱导分化为树突状细胞(DCs)。
从13例CML患者中分离出骨髓单个核细胞(MNCs),从5名健康供者中分离出外周血MNCs。将分离出的MNCs与1000 U/ml的重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、500 U/ml的重组人白细胞介素-4(rhIL-4)和50 U/ml的肿瘤坏死因子-α(TNF-α)共培养10天。通过瑞氏染色、倒置显微镜和电子显微镜观察形态学特征。采用流式细胞术检测CD(80)、CD(86)、CD(83)、CD(1a)和HLA-DR的表达,采用荧光原位杂交(FISH)进行细胞遗传学分析。采用酶联免疫吸附测定(ELISA)法检测白细胞介素-12(IL-12)的浓度,并通过混合淋巴细胞反应(MLR)检测抗原呈递功能。
细胞因子培养后,观察到具有精细膜突起的典型树突状外观。CD(80)、CD(86)、CD(83)、CD(1a)和HLA-DR标志物以及刺激同种异体T细胞的能力显著上调。FISH证实DCs来源于白血病细胞,且CML DCs分泌IL-12的水平高于CML MNCs。CML来源的DCs与正常个体来源的DCs在形态和免疫表型表达上无差异。然而,在MLR检测中,CML患者来源的DCs活性低于正常个体。
通过细胞因子共培养可将CML细胞诱导分化为白血病DCs。