猪外周血及骨髓源树突状细胞分化方法的建立
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摘要
本研究对猪源树突状细胞(dendritic cell,DC)的分化条件进行了摸索,通过对试验猪只日龄的选择、刺激物浓度和刺激时间的比较以及DC表面分子标记的选择,确定了猪外周血和骨髓源DC的分化条件。静脉采集15日龄猪只抗凝血分离获得外周血单核细胞(peripheral blood mononuclear cells,PBMCs),贴壁12 h后,用终浓度80 ng/mL的GM-CSF和40 ng/mL的IL-4共同刺激7 d,用CD152作为DC的表面分子标志,试验结果显示:贴壁的单核细胞在GM-CSF和IL-4的共同刺激下,7 d后能够将超过60%的单核细胞刺激分化为CD152~+的DC。取15日龄猪只的股骨和胫骨并冲洗骨髓腔,参考外周血来源DC的分化方法,结果显示:可以获得约63%的骨髓源单核细胞刺激分化为CD152~+的DC。本研究建立的DC分化方法为进一步研究病毒感染后猪体建立获得性免疫应答的机制奠定了平台基础。
As the main antigen-presenting cells, dendritic cells(DCs) play a key role in the induction of adaptive immunity. In order to develop a suitable method for differentiation of DCs from porcine peripheral blood and bone marrow, the factors evaluated in this study included piglet age, stimulator concentration, stimulation time and cell surface molecular markers. The results showed that 15-day-old piglets were suitable for isolation of peripheral blood mononuclear cells(PBMCs) and DC differentiation. The PBMCs isolated from piglet blood were cultured in six well plates with 2×106 cells per well. After 12 hours adherence, 80 ng/mL of GM-CSF and 40 ng/mL of IL-4 were added and stimulation was allowed for 7 days and CD152 was used as the surface molecular marker. The results showed that under the above condition about 60% of monocytes differentiated into CD152~+ cells, and under the above condition about 63% of bone marrow derived cell differentiated into CD152~+ cells. The development of porcine DC differentiation methods from PBMCs and bone marrow provided a useful tool for the further study on antigen presenting in virus infection and immune protection.
As the main antigen-presenting cells, dendritic cells(DCs) play a key role in the induction of adaptive immunity. In order to develop a suitable method for differentiation of DCs from porcine peripheral blood and bone marrow, the factors evaluated in this study included piglet age, stimulator concentration, stimulation time and cell surface molecular markers. The results showed that 15-day-old piglets were suitable for isolation of peripheral blood mononuclear cells(PBMCs) and DC differentiation. The PBMCs isolated from piglet blood were cultured in six well plates with 2×106 cells per well. After 12 hours adherence, 80 ng/mL of GM-CSF and 40 ng/mL of IL-4 were added and stimulation was allowed for 7 days and CD152 was used as the surface molecular marker. The results showed that under the above condition about 60% of monocytes differentiated into CD152~+ cells, and under the above condition about 63% of bone marrow derived cell differentiated into CD152~+ cells. The development of porcine DC differentiation methods from PBMCs and bone marrow provided a useful tool for the further study on antigen presenting in virus infection and immune protection.
引文
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[25]Hu Y,Cong X,Chen L,et al.Synergy of TLR3 and 7ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/My D88-NF-κB signaling pathway[J].Sci Rep,2016,6:23977.
[2]Steinman R M,Cohn Z A.Identification of a novel cell type in peripheral lymphoid organs of mice.I.Morphology,quantitation,tissue distribution[J].J Exp Med,1973,137(5):1142-1162.
[3]Steinman R M.Decisions about dendritic cells:past,present,and future[J].Annual Rev Immunol,2012,30:1-22.
[4]Merad M,Sathe P,Helft J,et al.The dendritic cell lineage:ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting[J].Annu Rev Immunol,2013,31:563-604.
[5]Macri C,Pang E S,Patton T,et al.Dendritic cell subsets[J].Semin Cell Dev Biol,2018,84:11-21.
[6]Banchereau J,Steinman R M.Dendritic cells and the control of immunity[J].Nature,1998,392(6673):245-252.
[7]Gregori S.Dendritic cells in networks of immunological tolerance[J].Tissue Antigens,2011,77(2):89-99.
[8]Bancherau J,Paczesny S,Blanco P,et al.Dendritic cells:controllers of the immune system and a new promise for immunotherapy[J].Ann N Y Acad Sci,2003,987:180-187.
[9]Helft J,Anjos-Afonso F,van der Veen A G,et al.Dendritic cell lineage potential in human early hematopoietic progenitors[J].Cell Rep,2017,20(3):529-537.
[10]Collin M,Bigley V.Human dendritic cell subsets:an update[J].Immunology,2018,154(1):3-20.
[11]Guilliams M,Ginhoux F,Jakubzick C,et al.Dendritic cells,monocytes and macrophages:a unified nomenclature based on ontogeny[J].Nat Rev Immunol,2014,14(8):571-578.
[12]Villani A C,Satija R,Reynolds G,et al.Single-cell RNA-seq reveals new types of human blood dendritic cells,monocytes,and progenitors[J].Science,2017,356(6335).pii:eaah4573.
[13]See P,Dutertre C A,Chen J,et al.Mapping the human DC lineage through the integration of high-dimensional techniques[J].Science,2017,356(6342).pii:eaag3009.
[14]Satpathy A T,K C W,Albring J C,et al.Zbtb46expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages[J].JExp Med,2012,209(6):1135-1152.
[15]Inaba K,Swiggard W J,Steinman R M,et al.Isolation of dendritic cells[J].Curr Protoc Immunol,2001,Chapter 3:Unit 3.7.
[16]Wang S,Sun X,Zhou H,et al.Interleukin-4 affects the mature phenotype and function of rat bone marrowderived dendritic cells[J].Mol Med Rep,2015,12(1):233-237.
[17]Scheicher C,Mehlig M,Zecher R,et al.Dendritic cells from mouse bone marrow:in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor[J].J Immunol Methods,1992,154(2):253-264.
[18]Inaba K,Inaba M,Romani N,et al.Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor[J].J Exp Med,1992,176(6):1693-1702.
[19]Burgess A W,Metcalf D.The nature and action of granulocyte-macrophage colony stimulating factors[J].Blood,1980,56(6):947-958.
[20]Poulin L F,Salio M,Griessinger E,et al.Characterization of human DNGR-1+BDCA3+leukocytes as putative equivalents of mouse CD8alpha+dendritic cells[J].J Exp Med,2010,207(6):1261-1271.
[21]Shimizu M,Yamada S,Kawashima K,et al.Changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome(PRRS)virus[J].Vet Immunol Immunopathol,1996,50(1-2):19-27.
[22]虞凌雪.表达猪GM-CSF重组PRRSV弱毒疫苗株的构建及其免疫特性分析[D].北京:中国农业科学院,2014.
[23]Park J Y,Kim H S,Seo S H,et al.Characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells[J].J Microbiol Biotechnol,2008,18(10):1709-1716.
[24]Chen X,Bai J,Liu X,et al.Nsp1αof porcine reproductive and respiratory syndrome virus strain BB0907 impairs the function of monocyte-derived dendritic cells via the release of soluble CD83[J].J Virol,2018,92(15).pii:e00366-18.
[25]Hu Y,Cong X,Chen L,et al.Synergy of TLR3 and 7ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/My D88-NF-κB signaling pathway[J].Sci Rep,2016,6:23977.