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

Abstract

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

CLC Number:

S852.4

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.

References

[1] HOTBLACK A,SESHADRI S,ZHANG L,et al.Dendritic cells cross-present immunogenic lentivector-encoded antigen from transduced cells to prime functional T cell immunity[J].Molecular Therapy,2017,25(2):504-511.
[2] LANZAVECCHIA A,SALLUSTO F.Regulation of T cell immunity by dendritic cells[J].Cell,2001,106(3):263-266.
[3] FERNANDO E D,OCHOA V,MERLOTTI A,et al.Extracellular acidosis and mTOR inhibition drive the differentiation of human monocyte-derived dendritic cells[J].Cell Reports,2020,31(5):107613-107627.
[4] 郎需龙,王兴龙,孙春辉,等.IL-10对感染口蹄疫病毒的树突状细胞的影响[J].中国畜牧兽医,2009,36(11):27-29. LANG X L,WANG X L,SUN C H,et al.The effects of IL-10 on dendritic cells infected by Foot-and-mouth virus[J].China Animal Husbandry & Veterinary Medicine,2009,36(11):27-29.(in Chinese)
[5] HEMANN E A,GREEN R,TURNBULL J B,et al.Interferon-λ modulates dendritic cells to facilitate T cell immunity during infection with influenza A virus[J].Nature Immunology,2019,20(8):1035-1045.
[6] CONSTANTINO J,GOMES C,FALCAO A,et al.Dendritic cell-based immunotherapy:A basic review and recent advances[J].Immunologic Research,2017,65(4):798-810.
[7] 赵良中,李瑶,张铎,等.绿色荧光蛋白标记的树突状细胞对体外培养的自然杀伤细胞的影响[J].中国畜牧兽医,2012,39(8):139-141. ZHAO L Z,LI Y,ZHANG D,et al.Effects of GFP labeled dendritic cells on natural killer cells cultured in vitro[J].China Animal Husbandry & Veterinary Medicine,2012,39(8):139-141.(in Chinese)
[8] MACRI C,PANG E S,PATTON T,et al.Dendritic cell subsets[J].Seminars Cell and Development Biology,2018,84:11-21.
[9] ZHAO S,GAO Q,QIN T,et al.Effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen[J].Veterinary Microbiology,2014,171(1-2):74-86.
[10] 刘芳,乔艳艳,薛洋,等.猪外周血单核细胞来源的树突状细胞体外诱导培养[J].江苏农业学报,2016,32(3):588-592. LIU F,QIAO Y Y,XUE Y,et al.Porcine peripheral blood monocyte-derived dendritic cells cultured in vitro[J].Jiangsu Journal of Agricultural Sciences,2016,32(3):588-592.(in Chinese)
[11] VAN D L,COFFER P,WOLTMAN A M.Regulation of dendritic cell development by GM-CSF:Molecular control and implications for immune homeostasis and therapy[J].Blood,2012,119(15):3383-3393.
[12] 廖娟红,林祥梅,贾广乐.小鼠髓源未成熟树突状细胞的分离与培养[J].中国畜牧兽医,2013,40(7):107-111. LIAO J H,LIN X M,JIA G L.Isolation and culture of mouse bone marrow-derived dendritic cells[J].China Animal Husbandry & Veterinary Medicine,2013,40(7):107-111.(in Chinese)
[13] NDIAYE M,WARNECKE A,FLYTZANI S,et al.Rat bone marrow-derived dendritic cells generated with GM-CSF/IL-4 or FLT3L exhibit distinct phenotypical and functional characteristics[J].Journal of Leukocyte Biology,2016,99(3):437-446.
[14] 王芳,牛冬,余旭平,等.猪生物医学模型研究进展[J].动物医学进展,2009,30(4):99-102. WANG F,NIU D,YU X P,et al.Progress on swine biomedical model[J].Progress in Veterinary Medicine,2009,30(4):99-102.(in Chinese)
[15] 马兵,吴军,郑峻松,等.猪骨髓树突状细胞的体外扩增及鉴定[J].第三军医大学学报,2002,24(3):255-257. MA B,WU J,ZHENG J S,et al.Amplification and identification of porcine bone marrow-derived dendritic cells in vitro[J].Journal of Third Military Medical University,2002,24(3):255-257.(in Chinese)
[16] 沈海燕,赵明秋,琚春梅,等.猪树突状细胞的体外培养及其主要免疫生物学特性研究[J].中国预防兽医学报,2010,32(5):388-393. SHEN H Y,ZHAO M Q,JU C M,et al.In vitro cultivation and characterization of dendritic cells from porcine peripheral blood monocytes[J].Chinese Journal of Preventive Veterinary Medicine,2010,32(5):388-393.(in Chinese)
[17] GAO Q,ZHAO S,QIN T,et al.Effects of Porcine epidemic diarrhea virus on porcine monocyte-derived dendritic cells and intestinal dendritic cells[J].Veterinary Microbiology,2016,179(3-4):149-158.
[18] LIN J,TU C,MOU C,et al.CpG DNA facilitate the inactivated Transmissible gastroenteritis virus in enhancing the local and systemic immune response of pigs via oral administration[J].Veterinary Immuno-logy & Immunopathology,2016,172:1-8.
[19] MOU C,ZHU L,XING X,et al.Immune responses induced by recombinant Bacillus subtilis expressing the spike protein of transmissible gastroenteritis virus in pigs[J].Antiviral Research,2016,131:74-84.
[20] STEINMAN R M,COHN Z A.Identification of a novel cell type in peripheral lymphoid organs of mice-Ⅰ.Morphology,quantitation,tissue distribution[J].The Journal of Experimental Medicine,1973,137(5):1142-1162.
[21] BARBOZA B A,FONSECA B P,VIOLA J P.NFAT1 transcription factor in dendritic cells is required to modulate T helper cell differentiation[J].Immuno-biology,2014,219(9):704-712.
[22] CARRASCO C P,RIGDEN R C,SCHAFFNER R,et al.Porcine dendritic cells generated in vitro:Morphological,phenotypic and functional properties[J].Immunology,2001,104(2):175-184.
[23] ARDAVIN C.Origin,precusors and differentiation of mouse dendritic cells[J].Nature Review Immunology,2003,3(7):582-590.
[24] 张东辉,侯敏,陈莹莹,等.Flt3L刺激体外培养小鼠骨髓源CD8α⁺树突状细胞及其成熟状态分析[J].中国病原生物学杂志,2014,9(1):33-36. ZHANG D H,HOU M,CHEN Y Y,et al.Ex vivo FLT3L stimulation of CD8α⁺ dendritic cells derived from mouse bone marrow and analysis of their maturity[J].Journal of Pathogen Biology,2014,9(1):33-36.(in Chinese)
[25] SUMMERFIELD A,MCCULLOUGH K C.The porcine dendritic cell family[J].Developmental and Comparative Immunology,2009,33(3):299-309.
[26] BERTRAM E,DAWICKI W,WATTS T H.Role of T cell costimulation in anti-viral immunity[J].Seminars of Immunology,2004,16(3):185-196.
[27] BIMCZOK D,SOWA E N,FABER-ZUSCHRATTER H,et al.Site-specific expression of CD11b and SIRPalpha (CD172a) on dendritic cells:Implications for their migration patterns in the gut immune system[J].European Journal of Immunology,2005,35(5):1418-1427.
[28] 李焕荣,王振生,隋丽华,等.猪皮肤源树突状细胞的分离和鉴定[A].第四届中国畜牧科技论坛论文集[C].2009. LI H R,WANG Z S,SUI L H,et al.Isolation and identification of swine skin-derived dendritic cells[A].Proceedings of the Fourth China Animal Husbandry Sci-tech Forum[C].2009.(in Chinese)

Expression of Porcine Colony-stimulating Factor and Interleukin-4 and Its Function in Inducing Porcine Dendritic Cells in vitro

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	CHEN Kaiwen, ZHAO Yunjiao, WANG Lei, ZHANG Jianbo, MA Yuhong, WU Guofang. Transcriptome Analysis of Porcine Leydig Cells Under Different Oxygen Concentrations [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2185-2195.
[2]	CAO Qi, JIN Qing, YUAN Yi, FENG Yixue, KONG Danni, CHEN Xianghong, WU Bin, SONG Wenbo, TANG Xibiao. Epidemiological Investigation of Common Respiratory Bacterial Pathogens in Large-scale Pig Farms [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2439-2449.
[3]	SHI Qiuying, DUAN Baomin, LI Ying, NIU Dengyun, GENG Chenyang, ZHOU Jiawei, WANG Rui, FENG Jingjing. Analysis of Genetic Variation of S1 Gene of Porcine Epidemic Diarrhea Virus in Some Areas of China from 2020 to 2021 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2468-2478.
[4]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[5]	LI Mingguo, LI Qingchun, CHEN Xin, LU Shihao, HE Fan, QI Mengfan, ZHANG Huapeng, REN Yujun, ZHANG Qingze, FU Binbin, XU Mengsi, AI Zikai, YAN Kun, FENG Yun, HUA Zaidong, HUANG Tao, BI Yanzhen. Effects of TET Demethylase on Porcine Oocyte Development [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1907-1917.
[6]	KANG Pu, REN Zhaowen, CAI Rujian, LYU Zongji, HUANG Yuhao, WANG Xiaohu. Variation Regularity and Transmission of PRRSV-2 Lineage 8 Strains in Guangdong and Surrounding Provinces [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2001-2011.
[7]	JIANG Haijun, WANG Dan, GUO Yu, WEI Li, HOU Lei, WANG Jing, ZHU Shanshan, LIU Jue. Fusion Expression and Immunogenicity of Recombinant Cap-flagellin Protein of Porcine Circovirus Type 3 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2044-2052.
[8]	CHEN Tianbao, WANG Yani, LIU Baoling, WANG Gang, WANG Xiaohu, LIU Xuhui, CAI Rujian, HE Dongsheng. Research Progress on the Anti-Porcine Reproductive and Respiratory Syndrome Virus Effect and Mechanism of Traditional Chinese Medicine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2114-2129.
[9]	HUO Mingkai, GUAN Feihu, QIU Runhui, WEI Chunyan, YU Haitao, ZHU Jiale, ZHANG Hui. Study on the Mechanism of DDX1 Inhibiting Porcine Epidemic Diarrhea Virus Replication [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1556-1566.
[10]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[11]	ZHANG Guoqing, JIANG Yuhang, ZHANG Shuang, LI Letian, HAO Jiayi, CHEN Jing, ZOU Wancheng, LI Chang, LI Taiyuan, GAO Xu, JIN Ningyi. Preparation and Identification of PDCoV RBD Protein Peptide Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1575-1583.
[12]	LIU Xinyue, LI Lifang, WANG Zhenbao, ZHANG Wenliang, ZHANG Xin, ZHAO Hongtao, LI Pengyu, ZHENG Haotian, XIAO Jin, QI Peng. Adjuvants Screening and Immune Protection of Porcine Circovirus Type 2 Synthetic Peptide Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1068-1080.
[13]	ZHANG Jingxu, XU Yu, LIANG Haiying, ZENG Zhiyong, TANG Deyuan, WANG Bin, XU Songping, WAN Juan, ZHU Yang. Phylogenetic Evolution and Epitope Analysis of Pseudorabies Virus Guizhou Isolates and Vaccine Strains [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1093-1106.
[14]	LI Huawei, GUO Kewei, WANG Xuying, ZHANG Xiaoyu, LU Ziwei, LI Xinfeng, MA Hui, HOU Wenjing. Eukaryotic Expression and Subcellular Localization of Nonstructural Protein 7 of Porcine Reproductive and Respiratory Syndrome Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1160-1168.
[15]	MU Yijiao, YU Ruiming, ZHANG Liping, WANG Yonglu. Establishment and Application of ELISA for Detection of IgA Antibody Based on the Recombinant Full-length S2 Protein of Porcine Epidemic Diarrhea Virus Variant Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1185-1194.