小鼠骨髓源半成熟树突状细胞的培养与初步鉴定

Culture and Identification of Mouse Myeloid Semimature Dendritic Cells

目的探索培养与鉴定小鼠骨髓来源半成熟树突状细胞（smDC）的可行方法。方法分离、纯化6周龄C57BL/6小鼠骨髓单核细胞,以含10%胎牛血清、2 ng/ml重组小鼠粒细胞-巨噬细胞集落刺激因子（GM-CSF）和20ng/ml重组小鼠白细胞介素（IL）-4的RPMI-1640培养基培养9 d;再以肿瘤坏死因子-α（TNF-α）（40 ng/ml）刺激24 h获得smDC,同时以脂多糖（1μg/ml）刺激和不加刺激培养24 h获得成熟DC（mDC）和未成熟DC（iDC）。扫描电子显微镜、流式细胞术、ELISA检测iDC、smDC和mDC的形态、细胞表面标志及细胞因子的表达。建立体外淋巴细胞反应模型,采用细胞计数试剂盒检测smDC对异基因淋巴细胞的活化作用。结果smDC的体积介于iDC和mDC之间、多呈类圆形或圆形,胞体直径约为15μm,树突长度多在5-10μm。smDC、iDC与mDC均高表达CD11c,smDC表达CD40、CD80、CD86及MHC-Ⅱ介于iDC和mDC之间。smDC分泌IL-1β、IL-6和IL-12显著低于mDC（P〈0.01）、分泌IL-12显著低于iDC（P〈0.05）,分泌IL-1β、IL-6与iDC比较差异无显著性（P〉0.05）;smDC、iDC与mDC分泌IL-10差异无显著性（P〉0.05）。smDC对异基因淋巴细胞激活作用显著低于mDC和阳性对照（P〈0.01）,而与阴性对照和iDC差异无显著性（P〉0.05）。结论smDC可能是一个功能和形态上相对独立的DC亚群,体外利用GM-CSF、IL-4和TNF-α诱导骨髓单核细胞是获得smDC的有效方法。

Objective To investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell（smDC） in vitro.Methods Myeloid monocytes derived from 6-week-old C57BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum,2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor（GM-CSF）,and 20 ng/ml recombinant murine interleukin（IL）-4 for 9 days.Then cells were incubated with 40 ng/ml tumor necrosis factor-α（TNF-α） for 24 hours to obtain smDC.Meanwhile,smDC was differentiated into mature dendritic cell（mDC） or immature dendritic cell（iDC） by treatment with 1μg/ml lipopolysaccharide（LPS） or without LPS.The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy.Surface markers such as CD11c,CD40,CD80,CD86,and MHC-Ⅱ were tested by flow cytometry.IL-1β,IL-6,IL-12,and IL-10 in thesupernatant were tested by ELISA.The activation of allogene lymphocyte（BALB/c mice） stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-8 in vitro.Results The shape of smDC was round or oval-shaped,and the diameter of smDC was about 15μm.The length of smDC dendrite was between 5 to 10μm.smDC,iDC,and mDC all expressed high level of CD11c.The expressions of MHC-Ⅱ,CD40,CD80,and CD86 on smDC were higher than those of iDC and lower than those of mDC.IL-1β,IL-6,and IL-12 secretion of smDC was significantly lower than that of mDC（P〈0.01）,and IL-12 was significantly lower than that of iDC（P〈0.05）,while no significant difference of IL-1β and IL-6 secretion was found between smDC and iDC（P〉0.05）.Furthermore,IL-10 secretion was not significantly different among these three kinds of DCs（P〉0.05）.The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control（P〈0.01）,but had no significant difference when compared with that of iDC and negative control（P〉0.05）.Conclusions smDC may be a relatively independent dendritic cell sub-population in terms of function and morphology.It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF,IL-4,and TNF-α in vitro.