小鼠骨髓源CD103 ＋DC分离培养及LPS对其形态与功能特征的影响

In vitro culture of CD103^+ DCs from mouse bone marrow and effects of LPS on its morphology and functional characteristics

旨在建立C57BL/6小鼠骨髓源CD103^+树突状细胞(CD103^+dendritic cell,CD103^+DC)分离培养方法,阐述LPS对其形态与功能特征的影响。在无菌条件下分离C57BL/6小鼠骨髓细胞,并用重组GM-CSF和FLT3L对其进行体外联合诱导培养;利用光镜、扫描电镜、荧光显微镜和流式细胞术,分别对LPS作用前后细胞形态、表型及功能进行了分析。结果表明,细胞培养至第3天有零星集落出现,第13天后集落开始分散,可见典型的树突状突起,第15天后可得到大量的CD103^+DC,加LPS刺激培养24 h后细胞表面树突样结构更加明显;分离培养的骨髓细胞能够表达表面分子CD103,其表达率达90%以上。RPMI-1640组(LPS未刺激组)可吞噬VOA的CD103^+DC比例为25.70%,能够表达MHC-Ⅱ和CD83阳性细胞分别为41.31%和13.79%;LPS刺激组可吞噬VOA的CD103^+DC比例为10.33%,能够表达MHC-Ⅱ和CD83的阳性细胞分别为68.10%和24.71%。MTT法检测结果显示,经LPS处理的CD103^+DC刺激T细胞增殖的能力明显增强。综上所述,分离于C57BL/6小鼠的骨髓细胞,可在体外经FLT3L和GM-CSF共同诱导培养出CD103^+DC,LPS可促进CD103^+DC的成熟。

In order to establish a method of inducing and culturing CD103 ＋ dendritic cells （CD103 ＋ DCs） from C57BL/6 mice bone marrow in vitro ,and investigate the effects of LPS on their morphology and functional feature. The bone marrow cells （BMCs） were isolated and cultured in the presence of recombinant granulocyte-macrophage colony-stimulating factor （GM-CSF） and fms-like tyrosine kinase 3 ligand （FLT3L）, and then observed the effect of LPS on their morphology, phenotype and function. The results showed that the modality of BMCs diversified after being cultured in GM-CSF and FLT3L for 3 d, and grew clustered-liked.As time went on, the cell colonies became larger and larger. When culturing for 13 d, the BMC colonies started separating, typical morphology with dendritic processes could be observed. A number of morphologically typical dendritic cells were observed after culturing for 15 d. The dendritic structure became more obvious after BMCs were treated with LPS for 24 h under scan electron microscope. The induced BMCs were able to express CD103 on the cell surface, and the positive rate of CD103 ＋DC was over 90%. The percentage of phagocytosis of CD103 ＋ DCs was 25.70% in the group of RPMI-1640, but the percentage of phagocytosis in the group of LPS dropped to 10.33%. In the group of RPMI-1640, 41.31% CD103 ＋DCs expressed MHC-II, 13.79% expressed CD83. The CD103 ＋DCs treated by LPS showed a higher MHC-II expression rate of 68.10%, and increase expression rate in CD83 （24.71%）. MTT assay showed that the ability of CD103 ＋DC treated with LPS to stimulate the proliferation of the naive allergenic T cells was stronger than those CD103 ＋DC untreated with LPS. In conclusion, a large number of dendritic cells can be generated by culturing BMCs from C57BL/6 mice in vitro ,and LPS shows positive effect on CD103 ＋ DC mature.