Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression
Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression
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DOI：
10.1016/j.jim.2008.07.012
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发表时间：
2008-11-30
影响因子：
2.2
通讯作者：
Foucras, Gilles
中科院分区：
文献类型：
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作者：
Foulon, Eliane;Foucras, Gilles
尽管对啮齿动物树突状细胞的研究主要依赖于骨髓来源的树突状细胞（bm - dc），但没有关于绵羊树突状细胞的数据，绵羊是一个主要用于免疫学和移植研究的物种。我们开发了一种培养方案，使用从杆状病毒感染的昆虫细胞中纯化的6x（His）标记的重组GM-CSF来生产羊bm - dc。在重组GM-CSF存在下培养羊骨髓祖细胞，6-7天后可产生大量CD11 c阳性细胞。采用流式细胞术和电镜技术对脑转移型树突细胞进行表型观察。鉴定出两个DC亚群表达不同水平的MHC 11类分子，受体介导的内吞作用不同，并且可以通过CD11b表达分离。当分离的细胞与微生物产物一起孵育时，它们与其他物种中被认为是TLR2和TLR4激动剂的细胞反应不同。事实上，尽管CD11b（int/hi）细胞对脂质胆酸或脂多糖诱导的成熟具有部分抗性，但在CD11b（dull）细胞上观察到MHC II类上调。此外，在异体反应中，这些细胞对CD4 T细胞有很强的刺激能力。该方案将有助于分析绵羊DC与病原体的相互作用，并使未来的疫苗开发研究成为可能。(C) 2008 Elsevier B.V.版权所有
Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11 c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class 11 molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b(int/hi) cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b(dull) cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines. (C) 2008 Elsevier B.V. All rights reserved.