α-干扰素联合GM-CSF体外诱导培养脐血树突状细胞及其功能的测定

The Induction and Function of Dendritic Cells from Human Cord Blood with IFN-α and GM-CSF in vitro

目的 探讨重组人α-干扰素 (rh IFN-α)联合重组人粒 -巨噬细胞集落刺激因子 (rh GM- CSF)体外诱导培养脐血树突状细胞 (DCs)的实验方法。方法 人脐血贴壁单个核细胞加入 rh IFN- α及 rh GM- CSF诱导培养 ,镜下观察培养细胞形态 ;流式细胞术检测培养细胞 CD83、CD86的表达 ;MTT法检测诱导培养的 DCs体外刺激同种异体 T淋巴细胞增殖的活性。结果 人脐血贴壁单个核细胞经 rh IFN-α联合 rh GM- CSF共同培养后 ,第 7d镜下可见有表面呈树状突起的 DCs;细胞免疫表型检测结果示培养细胞有成熟 DCs特异性标记 CD83及共刺激分子CD86表达 ;rh IFN -α为 6 0 0 U / m l、rh GM- CSF为 2 0 0 0 U / ml时为脐血 DCs诱导培养的合适浓度 (培养细胞 CD83+CD86 + 细胞比率最高 ) ;以诱导培养 7d的脐血细胞 (含 DCs)作为刺激细胞与同种异体 T淋巴细胞 (反应细胞 )以不同比例混合培养时 ,DCs均可刺激其增殖 ,其中以反应细胞∶刺激细胞为 2 0∶ 1时最明显。结论 rh IFN- α联合rh GM- CSF细胞因子体外诱导人脐血贴壁单个核细胞 ,可生成具有典型形态及功能的成熟

Objective To probe into the possibility of obtaining dendritic cells(DCs) from human cord blood with rhIFN-α and rhGM-CSF. Methods The plastic adherent cells (monocyte-rich cells) from cord blood were cultured with rhGM-CSF and rhIFN-α for 7 days.The morphological properties of DCs were observed by microscopy, the expression of CD83 and CD86 on DCs were determined by flow cytometry.The function of DCs stimulating allogeneic T lymphocyte proliferation was detected by MTT colorimetric method. Results After 7 days culture, some of the cultured cells acquired typical DC morphology and showed increased expression of CD83 and CD86.The yield of DCs attained its peak when the concentrations of rhIFN-α and rhGM-CSF were 600U/ml and 2000 U/ml, respectively. When cultured cells (containing DCs) as stimulator cells were co-cultured with allogeneic T lymphocytes (effector cells) in different ratio, they could stimulate T cell proliferation remarkably, especially when the ratio of effector cells to stimulator cells was 20 to 1. Conclusion Our findings suggest that cord blood adherent cells, when cultured with rhGM-CSF and rhIFN-α, can be induced into mature DCs with the function of stimulating allogeneic T cell proliferation.