黄芪多糖体外诱导脐血单核细胞分化为树突状细胞及其免疫学特征

APS on Inducing the Effect of Differentiating Cord Blood Monocyte into the Dendritic Cells Induced by Astragalus Polysaccharides In Vitro and its Immunological Characteristic.

目的观察黄芪多糖(astragaluspolysaccharides,APS)代替细胞因子在体外对脐血单核细胞向树突状细胞分化的作用及细胞免疫学特征的变化。方法无菌条件下采集脐血,用淋巴细胞分离液分离获得脐血单个核细胞。将获得的单核细胞分为3组,实验组:在含有黄芪多糖(浓度为100mg/L)的10%胎牛血清的RPMI-1640完全培养液中培养;阴性对照组:在未加黄芪多糖的RPMI-1640完全培养液中培养;阳性对照组:在含有细胞因子白介素4(IL-4)、粒单细胞集落刺激因子(GM-CSF)和肿瘤坏死因子-α(TNF-α)的RPMI-1640完全培养液中培养;培养过程中用倒置光学显微镜和扫描电镜观察细胞形态;收集部分培养第12天的细胞利用流式细胞仪检测各组细胞表面CD1a、CD80、CD83和CD86分子的表达。结果在培养的第72h后实验组和阳性对照组细胞形态开始变化,随着培养时间的延长,树突状结构更加明显,第12天细胞呈典型的树突状细胞形态;阴性对照组细胞生长缓慢,细胞无成簇生长,培养至第12天细胞呈梭形巨噬细胞形态。培养至10天的实验组细胞扫描电镜下可见细胞表面粗糙,胞体突起成不规则形态,突起的长短、粗细、薄厚不等。培养12天后实验组、阳性对照组细胞别高表达DCs特异性抗原CD1a、CD80、CD83和CD86,与对照组对应比较差异均有显著性(P<0.01);实验组与阳性对照组之间比较差异无统计学意义(P>0.05)。结论黄芪多糖及细胞因子体外均可诱导脐血单核细胞(DCs前体细胞)定向分化为功能性(成熟)DCs。

Objectives To elucidate the effect of APS replacing cytokine on inducing the cord blood monocyte in vitro into the dendritic cells （DCs） and its cellar immunological characteristic. Methods ①The cord blood monocytes were isolated and obtained by lymphocyte isolation, three groups were divided： ②Cultured in the RPMI - 1640 culture with GM - CSF/IL -4/TNF - α, as the positive control group, with APS in concentration （100mg/L） as the experimental group, and without GM - CSF/IL -4/ TNF -α and APS, as the negative control group, respectively. The morphotype of DCs was identified by inverted optical microscope or scanning electron microscope. The phenotype of cultured 12 days DCs （ CD1a, CD80, CD86, and CD83） was identified by flowcytometry. Results Cultured for 72 hours , the morphous of cell of the experimental group grew clustering and began to change from round to irregularity, appearing rough cell face and barb pustute. The longer cell cultured, the more obvious the dendritic structure is. The experimental group cell cultured for 12 days had the most typical dendritic structure, the negative control group cell had no dendritic structure and became the macrophage when cultured for 12 days. The experimental group cell cultured for 10 days showed typical dendritic morphotype by SEM. The experiment group cell and the positive group cell cultured for 12 days significantly expressed the high level phenotype of DCs（ （CD1a, CD80, CD86, and CD83））by flowcytometry. Conclusions APS and cytokine both could induce the cord blood monocyte to direofive differentiate into functional DC.