骨髓单个核细胞分化为CD103^+树突状细胞的方法研究（英文）

Methods of CD103^+ dendritic cells differentiation from bone marrow

目的:探索小鼠骨髓单个核细胞在体外培养诱导其分化为CD103^+树突状细胞(DCs)的方法,为免疫性疾病的防治提供实验基础。方法:分离小鼠胫腓骨,冲洗骨髓腔得到骨髓细胞,利用单个核细胞分离液分离出单个核细胞,加入粒细胞巨噬细胞集落刺激因子(GM-CSF)、Fms样酪氨酸激酶3配体(FLT3L)诱导其分化为成熟的DCs。显微镜下观察细胞大小、形态、分布,流式细胞术检测细胞CD103^+及其共刺激分子的表达水平,再利用磁珠分选出CD103^+DCs,检测其纯度。结果:单个核细胞培养8 d后,倒置显微镜下观察到细胞分化为多种形态,大部分呈圆形或不规则,大小不一,细胞周围有细小凸起,成簇状分布,细胞表面CD11c和CD103增多,表面标志物CD40、CD80、CD86和MHC-Ⅱ升高,磁珠分选纯化后CD103^+DCs纯度可达95.7%,分选后的CD103^+DCs可有效促进T细胞分化。结论:利用GM-CSF和FLT3L联合诱导法进行体外培养,能够得到纯度较高的CD103^+DCs。

Objective:This study was performed to explore methods of inducing and culturing CD103^+dendritic cells(DCs)from mouse bone marrow mononuclear in vitro.Methods:The bone marrow mononuclear cells were obtained from femurs and tibias,and purified by mononuclear cells separation solution.Cells incubated with granulocyte-macrophage colony-stimulating factor(GM-CSF)and Fms-like tyrosine kinase-3 ligand(FLT3L)were added to induce cell differentiation.The morphology,phenotype and distribution were observed under invert microscope.The expression of CD103^+DCs and co-stimulatory molecules were detected by flow cytometry.CD103^+DCs were sorted by magnetic beads and detected the purity.Results:After 8 days culture,the mononuclear cells were differentiated into various forms under the microscope.Most of them were round or irregular,various morphologies and distribute clustered with plenty of dendrites.The expression of CD11c,CD103 and co-stimulatory molecules elevated significantly,and the purity of CD103^+ DCs was 95.7%.Besides,the CD103^+DCs could stimulate T cell differentiation efficiently.Conclusion:CD103^+DCs can be efficiently induced by GM-CSF and FLT3L in vitro.