人外周血树突状细胞的诱导、鉴定及活性测定

Induction,identification and activity of human plasmacytoid dendritic cells

目的探讨人外周血来源单个核细胞诱导为树突状细胞(DC)的方法,并对诱导细胞进行活性测定。方法采用淋巴细胞分离液分离人外周血单个核细胞,然后加入100ng/ml人粒-巨噬细胞集落刺激因子和30ng/ml人白细胞介素4培养5d,隔天半量换液,补充半量的细胞因子,于第5、6天,每孔加入1000U/ml肿瘤坏死因子,于培养第7天收集悬浮细胞。倒置显微镜每天观察细胞生长情况,用CD1a和CD83通过流式细胞仪和免疫荧光染色检测细胞表面表型,把DC与T细胞混合培养,按1:1、1:2、1:5的比例加入T淋巴细胞,MTT法检测T细胞的增殖活性。采用单因素方差分析,分析各实验组与对照组的细胞表型差异和刺激T淋巴细胞增殖活性能力比较。结果培养7d即可得到大量DC,显微镜下可见细胞体积增大、数量增多、形态不规则、表面大量突起,为典型的DC特征,免疫荧光染色可以看到CD1a和CD83高表达,培养4d,流式细胞检测显示CD1a阳性表达率达80.80﹪,CD83阳性表达率达19.5﹪,CD1a和CD83双阳性细胞达16.9﹪,3组表达阳性率间比较,差异均有统计学意义(F=695.293,P<0.01)。同时能刺激T细胞的增殖反应,其中DC数与T细胞数比为1:1时的刺激增强作用最强,吸光度值为0.74,活性比对照组增加3倍,差异有统计学意义(P值均<0.05)。结论通过体外添加细胞因子,成功诱导外周血单个核细胞为DC。

Objective To establish the method of inducing dendritic cells from monocytes from human peripheral blood, and evaluate the activity of induced dendritic cells. Methods Mononuclear cells were isolated from normal human peripheral blood, and cultured in medium with 100 ng/ml hGM-CSF. 30 ng/ml hIL-4 was added on the 4th day. During the culture, half of the media was replaced every two days. 1000 pYml INF was added on the 5th and 6th days respectively. The dendritic cells were harvested on the 7th day. The morphological characteristics were observed under inverted microscope everyday. The CDIa and CD83 of cell surface antigen of the dendritic cells were detected by flow cytometry （FCM） and immunofluorescence. T-lymphocytes proliferating activity stimulated by the dendritic cells were evaluated by MTT, in which dendritic cells were cultured with auto-mixed T-lymphocyte in the rate of 1：1,1：2 and 1：5 respectively. Results After 7 days in induction culture, dendritic cells with high purity were generated. The quintessential characteristics of dendritic cells, including large cell size and rapidly increasing capacity, irregular shape and dendrites on the cells surface, were observed under light-microscope. Immunofluorescence staining revealed high expression of CDla and CD83, and FCM showed the percentage of CDla＋ cell was 80.80%, CD83＋ cell