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目的 建立体外诱导、扩增具有抗成熟特性的未成熟树突状细胞 (DC)的方法。方法 从健康成人外周血分离单个核细胞 (PBMC) ,以rhGM -CSF和rhIL -4体外培养 9d ,于第 7d加入rhIL -10 ,收集悬浮细胞 ,电镜观察形态 ,流式细胞仪测定细胞表型 ,并进行混合淋巴细胞反应 (MLR) ,观察其刺激同种异体未致敏T淋巴细胞增殖的能力。将培养的细胞分别用LPS、TNF -α继续刺激 2d ,再进行MLR ,观察结果。结果 PBMC经rhGM -CSF +rhIL -4诱导并经rhIL -10处理后获得的细胞具有典型的DC形态特征 ,表达DC的特异性标志CD1a ,不表达DC的成熟标志CD83 ,与对照组相比其共刺激分子CD86、CD40 的表达显著下降 ,MLR示不能刺激同种异体T细胞增殖。经LPS、TNF -α刺激后亦不能刺激T细胞增殖。结论 与rhIL -10联合培养可获得具有抗成熟特性的未成熟DC ,这对于诱导大面积深度烧伤病人的异体皮移植免疫耐受具有良好的应用前景。
Objective To establish a method for inducing and amplifying immature dendritic cells (DCs) with anti-mature characteristics in vitro. Methods Mononuclear cells (PBMCs) were isolated from peripheral blood of healthy adults. RhGM-CSF and rhIL-4 were cultured in vitro for 9 days and rhIL-10 was added on the 7th day. Suspension cells were collected and observed by electron microscopy. Cell phenotype was determined by flow cytometry , And mixed lymphocyte reaction (MLR) to observe its ability to stimulate allogenic non-sensitized T lymphocyte proliferation. The cultured cells were stimulated with LPS and TNF-α respectively for 2 days and then MLR. The results were observed. Results PBMCs induced by rhGM-CSF + rhIL-4 and treated with rhIL-10 had typical morphological features of DC, CD1a, which is the specific marker of DC, and CD83, which is not the maturation marker of DC. Compared with the control group Costimulatory molecules CD86, CD40 expression was significantly decreased, MLR shows that can not stimulate allogeneic T cell proliferation. After LPS, TNF-α stimulation can not stimulate T cell proliferation. Conclusion Immature DCs with anti-mature characteristics can be obtained by co-culture with rhIL-10, which has good application prospects for inducing allograft immune tolerance in patients with extensive deep burn.