健康人外周血树突状细胞的体外诱导及其功能分析

Functional analysis of dendritic cells from peripheral blood of the healthy people induced in vitro

目的分析以健康人AB血清与rhCD40L体外诱导健康人外周血树突状细胞（Dc）的功能。方法对健康人外周血单个核细胞进行体外培养，在以健康人AB血清为基础的培养体系中加入粒细胞巨噬细胞集落刺激因子（GM-CSF）、重组人白细胞介素（rhIL）-4、rhCDnoL等细胞因子，诱导单个核细胞分化形成DC，采用倒置显微镜及瑞特-吉姆萨染色观察，流式细胞术行DC表型鉴定，四甲基偶氮唑蓝比色（MTT）法进行混合淋巴细胞反应（MLR），检测其抗原刺激能力，酶联免疫吸附（ELISA）法检测DC培养上清IL-12的分泌。结果培养7d后的细胞具有典型的DC形态，并上调表达DC特征性表面分子CD83及共刺激分子CD40、CD80、CD86，第0、1、3、5、7天，5个时间点间CD83、CD40、CD80、CD86、CD14表达差异有统计学意义（F值分别为50．253、243．769、248．181、191．267、226．339，均P〈0．05）。培养后的DC可较强地刺激同种自体淋巴细胞增殖，GM-CSF加rhIL-4、rhCD40L组较GM—CSF加rhIL-4组刺激反应能力强。培养的DC自培养第5天始即有IL-12分泌，未加CD40L组IL-12p40分泌量为（42．92±1．54）Pg／ml，加CD40L组为（136．18±5．27）Pg／ml；培养第7天，IL-12p40分泌明显增多，两组分别为（60．09±2．27）pg／ml及（322．30±30．60）Pg／ml，差异有统计学意义（t=-44．941、-22．611，均P〈0．05）。结论健康人外周血单个核细胞可在以健康人AB血清与rhCD40L为主的培养体系中诱导成DC。

Objective To establish a method to induce dendritic cells （DC） from peripheral blood mononuclear cells of healthy people in normal human AB serum in vitro and to identify the phenotype and the function of DC. Methods Peripheral blood mononuclear cells （PBMNC） of healthy people were cultured in RPMI 1640 media including human AB serum, GM-CSF, rhIL-4, and/no rhCD40L for 7 days to generate DC, which were identified by morphological features, surface antigen expression and the ability to stimulate T cells. Results After cultured and induced, DC displayed typical morphology with elongated dendritic process viewed by inverse light microscope as well as Wright-Gimsa stain. Mature DC express CD83 and the costimulatory molecules CD40, CD80 and CD86 to effectively activate T cells. In the five time points of 0 day, 1st day, 3rd day, 5th day and 7th day, the expression of CD83, CD40, CD80, CD86 and CD14 were significantly different （F = 50.253, 243.769, 248.181, 191.267 and 226.339, respectively, P 〈 0.05）. The ability to stimulate T ceils in GM-CSF, rhIL-4, and rhCD40L group was also stronger than that in GM-CSF and rhlL-4 group. DC started to secrete IL-12 from 5th day, the values were （42.92±1.54） pg/ml and （136.18±5.27） pg/ml in group of plus CD40L and of non plus CD40L, respectively. The secretion of the two groups of IL-12 were （60.09±2.27） pg/ml and （322.30±30.60） pg/ml （t = -44.941, -22.611, bath P 〈 0.05）. There are significant differences between the two groups. Conclusion DCs can be cultured from the peripheral blood of healthy people in normal human AB and rhCD40L serum.