猪集落刺激因子和白细胞介素-4的真核表达及其体外诱导猪树突状细胞的研究

doi:10.16431/j.cnki.1671-7236.2021.03.024

摘要/Abstract

摘要： 试验旨在建立猪集落刺激因子（granulocyte-macrophage colony stimulating factor，GM-CSF）和白细胞介素-4（interleukin-4，IL-4）的真核表达体系，应用表达的2种细胞因子体外诱导猪树突状细胞并检测其细胞功能。首先构建GM-CSF和IL-4真核表达载体，并在HEK293细胞上进行表达验证；其次应用表达的2种细胞因子诱导猪骨髓源和血液源单核细胞；最后分别采用荧光显微镜、流式细胞术检测猪树突状细胞的表面标志CD1a、SLA-Ⅱ和SWC3a，并进行树突状细胞形态学和免疫学功能鉴定。结果显示，本研究构建的2种真核表达载体在HEK293细胞中成功表达GM-CSF和IL-4。形态学观察发现，细胞因子诱导的单核细胞培养3 d后有聚集现象，6 d时可观察到典型的树突状突起。流式细胞术检测发现，表达的细胞因子可成功诱导猪骨髓源和血液源单核细胞转化为树突状细胞，其SWC3a⁺/SLA-Ⅱ⁺和CD1a⁺/SLA-Ⅱ⁺双阳性比例显著提高，与商品化细胞因子处理组没有区别。细胞吞噬试验发现，表达的细胞因子诱导的树突状细胞为未成熟树突状细胞，具有很强的细胞吞噬能力。本试验成功建立了在真核细胞中表达猪重组蛋白GM-CSF和IL-4的方法，并联合应用2种重组细胞因子体外诱导获得猪骨髓源和血液源单核树突状细胞，为进一步研究猪树突状细胞与各种病原微生物作用奠定基础。

关键词: 猪; 集落刺激因子; 白细胞介素-4; 树突状细胞

Abstract: The purpose of the experiment was to establish a eukaryotic expression system for porcine colony-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4),and use the expressed two cytokines to induce porcine dendritic cells in vitro and test their cell function.Firstly,the eukaryotic expression vectors of GM-CSF and IL-4 were constructed and verified on HEK293 cells.Then,the two cytokines were used to induce porcine bone marrow-derived and blood-derived monocytes.Finally,the surface markers CD1a,SLA-Ⅱ and SWC3a of porcine dendritic cells were detected by fluorescence microscopy and flow cytometry respectively,and the morphological and immunological functions of dendritic cells were identified.The results showed that the two eukaryotic expression vectors successfully expressed GM-CSF and IL-4 in HEK293 cells.Morphological observation showed that monocytes induced by cytokines had aggregation after 3 days of culture,and typical dendritic processes could be observed at the 6th day.Flow cytometry showed that the expressed cytokines could successfully induce the transformation of porcine bone marrow-derived and blood-derived monocytes into dendritic cells,and the double positive rates of SWC3a⁺/SLA-Ⅱ⁺ and CD1a⁺/SLA-Ⅱ⁺ were significantly increased,and there was no difference from the commercial cytokine treatment group.The results of cell phagocytosis test showed that the dendritic cells induced by the expressed cytokines were immature dendritic cells with strong phagocytic ability.In this study,we successfully established a method for the expression of porcine recombinant protein GM-CSF and IL-4 in eukaryotic cells,and induced mononuclear dendritic cells from pig bone marrow and blood using two recombinant cytokines in vitro,which laid a foundation for further study on the role of porcine dendritic cells and various pathogenic microorganisms.

Key words: porcine; GM-CSF; IL-4; dendritic cells

中图分类号:

S852.4

涂冲智, 李昱辰, 林建. 猪集落刺激因子和白细胞介素-4的真核表达及其体外诱导猪树突状细胞的研究[J]. 中国畜牧兽医, 2021, 48(3): 1001-1012.

TU Chongzhi, LI Yuchen, LIN Jian. Expression of Porcine Colony-stimulating Factor and Interleukin-4 and Its Function in Inducing Porcine Dendritic Cells in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 1001-1012.

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