细粒棘球蚴抗原B对小鼠巨噬细胞RAW264.7的极化作用

doi:10.12140/j.issn.1000-7423.2023.01.004

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (1): 23-28.doi: 10.12140/j.issn.1000-7423.2023.01.004

细粒棘球蚴抗原B对小鼠巨噬细胞RAW264.7的极化作用

焦红杰¹(), 齐文静², 郭刚¹, 包建玲¹, 吴川川², 宋传龙¹, 李军¹, 张文宝¹^,², 严媚¹^,^*()

1.新疆医科大学第一附属医院，乌鲁木齐 830054
2.新疆医科大学基础医学院，乌鲁木齐 830054

收稿日期:2022-06-13 修回日期:2022-07-10 出版日期:2023-02-28 发布日期:2023-02-24
通讯作者: * 严媚（1967-），女，博士，教授，主任医师，从事儿科血液及肿瘤研究。E-mail：yan10mei25@163.com
作者简介:焦红杰（1987-），女，博士研究生，从事儿科血液及肿瘤研究。E-mail：374064774@qq.com
基金资助:
国家自然科学基金(82160031);国家自然科学基金(81830066);省部共建中亚高发病成因与防治国家重点实验室立项项目(SKL-HIDCA-2020-BC);新疆维吾尔自治区科技厅天山创新团队项目(2020D14027)

Polarization effect of Echinococcus granulosus antigen B on the mouse macrophage RAW264.7

JIAO Hongjie¹(), QI Wenjing², GUO Gang¹, BAO Jianling¹, WU Chuanchuan², SONG Chuanlong¹, LI Jun¹, ZHANG Wenbao¹^,², YAN Mei¹^,^*()

1. First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China
2. Basic Medical College of Xinjiang Medical University, Urumqi 830054, China

Received:2022-06-13 Revised:2022-07-10 Online:2023-02-28 Published:2023-02-24
Contact: * E-mail: yan10mei25@163.com
Supported by:
National Natural Science Foundation of China(82160031);National Natural Science Foundation of China(81830066);project of State Key Laboratory of Pathogenesis and Prevention of Middle Asian High Disease(SKL-HIDCA-2020-BC);Tianshan Innovation Team Project of Science and Technology Department of Xinjiang Uygur Autonomous Region(2020D14027)

摘要/Abstract

摘要：

目的探讨细粒棘球蚴抗原B（AgB）对巨噬细胞极化的调控作用。方法将RAW264.7巨噬细胞培养24 h后，分为M1、M2、AgB、AgB+M1、AgB+M2和空白对照组（M0组），每组3孔。待所有巨噬细胞贴壁3 h后，AgB、AgB+M1、AgB+M2组均加入羊源细粒棘球蚴囊液提取的天然AgB（终浓度为1 000 ng/ml），刺激1 h后，M1组和AgB+M1组加入脂多糖（LPS，终浓度为100 ng/ml）和γ干扰素（IFN-γ，终浓度为20 ng/ml）刺激分化20 h；M2组和AgB+M2组加入白细胞介素4（IL-4）、IL-13（终浓度均为20 ng/ml）刺激分化20 h；空白对照组不更换培养液，同步培养20 h。显微镜下观察巨噬细胞形态。提取各组巨噬细胞总RNA，RT-PCR检测刺激后巨噬细胞表面标志物精氨酸酶1（Arg-1）、肿瘤坏死因子α（TNF-α）的mRNA相对转录水平；蛋白质免疫印迹（Western blotting）分析巨噬细胞蛋白Arg-1、诱导型一氧化氮合酶（iNOS）的相对表达量；ELISA检测刺激后巨噬细胞培养上清中IL-10、TNF-α的表达变化。结果经刺激分化后，镜下可见M1组和AgB+M1组巨噬细胞大部分呈不规则形，有触角；M2组和AgB+M2组巨噬细胞大部分呈圆形或椭圆形，极少呈不规则形；M0组和AgB组巨噬细胞部分呈圆形、椭圆形，部分呈不规则形。RT-PCR结果显示，M2组和AgB+M2组巨噬细胞的Arg-1 mRNA相对转录水平分别为189.49 ± 68.43、435.83 ± 123.57（t = 246.30，P < 0.01），二者均高于M0组（1.00 ± 0.00）、M1组（1.87 ± 1.29）、AgB组（2.37 ± 2.06）、AgB+M1组（3.96 ± 1.92）（t = 188.50、187.60、187.10、185.50，均P < 0.01；t = 434.80、434.00、433.50、431.90，均P < 0.01）；M1和AgB+M1组巨噬细胞的TNF-α mRNA相对转录水平分别为8.34 ± 2.92、8.10 ± 1.54（t = 0.24，P > 0.05），二者均高于M0组（1.00 ± 0.00）、M2组（1.37 ± 0.64）、AgB组（2.86 ± 0.44）、AgB+M2组（1.62 ± 0.27）（t = 7.34、6.97、5.48、6.71，均P < 0.01；t = 7.10、6.74、5.24、6.48，均P < 0.01）。Western blotting检测结果显示，M2组巨噬细胞的Arg-1蛋白相对表达量为1.18 ± 0.35，高于M1组（0.33 ± 0.18）、AgB+M1组（0.58 ± 0.10）（t = 0.67、0.61，均P < 0.01），与AgB组（1.05 ± 0.17）、AgB+M2组（0.97 ± 0.27）比较差异无统计学意义（t = 0.20、0.13，均P > 0.05）；AgB+M2与M1组、AgB+M1组比较差异均有统计学意义（t = 0.52、0.48，均P < 0.05）。M1组和AgB+M1组iNOS蛋白相对表达量分别为0.95 ± 0.21、0.88 ± 0.02（t = 0.07，P > 0.05），二者均高于M0组（0.03 ± 0.00）、M2组（0）、AgB组（0）和AgB+M2组（0）（t = 0.92、0.95、0.95、0.95，均P < 0.01；t = 0.85、0.88、0.88、0.88，均P < 0.01）。ELISA检测结果显示，巨噬细胞培养上清液中，AgB+M1组和AgB+M2组IL-10细胞因子表达量分别为166.67 ± 56.67、213.33 ± 16.67，均高于M0组（0.00 ± 0.00）、M1组（43.33 ± 36.67）、M2组（50.00 ± 43.00）、AgB组（47.50 ± 25.00）（t = 166.70、123.30、116.70、119.20，均P < 0.05；t = 213.30、170.00、163.30、165.80，均P < 0.01）。M1、AgB+M1组TNF-α细胞因子表达量分别为833.13 ± 3.09、745.63 ± 118.00（t = 87.50，P > 0.05），均高于M0组（217.50 ± 32.26）、M2组（224.69 ± 17.68）、AgB组（308.44 ± 4.42）、AgB+M2组（251.25 ± 1.33）（t = 615.60、608.40、524.70、581.90，均P < 0.01；t = 528.10、520.90、437.20、494.40，均P < 0.01）。结论 AgB可上调巨噬细胞Arg-1的表达，使其向M2型方向极化，可能是宿主和寄生虫免疫的重要调控分子，参与巨噬细胞的免疫调节。

关键词: 细粒棘球绦虫, 分泌抗原, 抗原B, 巨噬细胞极化

Abstract:

Objective To investigate the regulatory effect of Echinococcus granulosus antigen B (AgB) on macrophage polarization. Methods After cultivated for 24 h, the RAW264.7 macrophages cells were designated to 6 groups: M1, M2, AgB, AgB+M1, AgB+M2 and blank control (M0), 3 wells each group. After all the cells attached to the well wall for 3 h, the AgB、AgB+M1、AgB+M2 group was respectively added with natural AgB extracted from sheep hydatid cyst fluid (1 000 ng/ml, final concentration); 1 h post-stimulation, the M1 and AgB+M1 group was respectively added with lipopolysaccharide (LPS, final concentration 100 ng/ml), and IFN-γ (20 ng/ml, final concentration) to stimulate differentiation for 20 h; M2 and AgB+M2 group was added with interleukin 4 (IL-4) and IL-13 (final concentration 20 ng/ml) to stimulate differentiation for 20 h; the control group was cultured in parallel without changing medium. The morphology of macrophage cells were observed microscopically. Total RNA of the macrophages in all groups was extracted for performing RT-PCR to detect the relative transcription levels of the surface markers on stimulated macrophages, including arginase 1 (Arg-1) and tumor necrosis factor α (TNF-α). The relative expression levels of Arg-1 and inducible nitric oxide synthase (iNOS) were analyzed by Western blotting. The change of IL-10 and TNF-α expression in the culture supernatant of stimulated macrophages were detected by ELISA. Results After stimulation and differentiation, most cells in the M1 group and AgB+M1 group were irregularly shaped and had antennae. The cells of M2 group and AgB+M2 group were mostly round or oval, and very few were irregular. The cells of M0 group and AgB group were partly round and oval, and partly irregular. RT-PCR showed that the relative transcription levels of Arg-1 mRNA in the M2 group and the AgB+M2 group were 189.49 ± 68.43 and 435.83 ± 123.57, respectively (t = 246.30, P < 0.01). They were higher than those in the M0 group (1.00 ± 0.00), M1 group (1.87 ± 1.29), AgB group (2.37 ± 2.06), AgB+M1 group (3.96 ± 1.92) (t = 188.50, 187.60, 187.10, 185.50, P < 0.01; t = 434.80, 434.00, 433.50, 431.90, all P < 0.01). The relative transcription levels of TNF-α mRNA in the M1 group and the AgB+M1 group were 8.34 ± 2.92 and 8.10 ± 1.54, respectively (t = 0.24, P > 0.05). They were higher than that of the M0 group (1.00 ± 0.00), M2 group (1.37 ± 0.64), AgB group (2.86 ± 0.44) and AgB+M2 group (1.62 ± 0.27) (t = 7.34, 6.97, 5.48, 6.71, P < 0.01; t = 7.10, 6.74, 5.24, 6.48, P < 0.01). Western blotting showed that the relative expression level of Arg-1 protein in the M2 group was 1.18 ± 0.35, which was higher than that in the M1 group (0.33 ± 0.18) and the AgB+M1 group (0.58 ± 0.10) (t = 0.67，0.61, P < 0.01). There was no significant difference on the Arg-1 protein relative expression level between the AgB group (1.05 ± 0.17) and the AgB+M2 group (0.97 ± 0.27) (t =0.20, 0.13, P > 0.05). There was statistical significance in AgB+M2 group compared with M1 group and AgB+M1 group (t = 0.52, 0.48, P < 0.05). The iNOS relative expression levels of M1 group and AgB+M1 group were 0.95 ± 0.21 and 0.88 ± 0.02 (t = 0.07, P > 0.05), respectively. They were higher than those in M0 group (0.03 ± 0.00), M2 group (0), AgB group (0) and AgB+M2 group (0) (t = 0.92, 0.95, 0.95, 0.95, P < 0.01; t = 0.85, 0.88, 0.88, 0.88, P < 0.01). The ELISA results showed that the expression level of IL-10 cytokine in the cell supernatant was 166.67 ± 56.67 in the AgB+M1 group and 213.33 ± 16.67 in the AgB+M2 group, respectively. They were higher than those in the M0 group (0.00 ± 0.00), M1 group (43.33 ± 36.67), M2 group (50.00 ± 43.00) and AgB group (47.50 ± 25.00) (t = 166.70, 123.30, 116.70, 119.20, all P < 0.05. t = 213.30, 170.00, 163.30, 165.80, P < 0.01). The expression levels of TNF-α cytokines in M1 group and AgB+M1 group were 833.13 ± 3.09 and 745.63 ± 118.00, respectively (t = 87.50, P > 0.05). They were higher than those in M0 group (217.50 ± 32.26), M2 group (224.69 ± 17.68), AgB group (308.44 ± 4.42), AgB+M2 group (251.25 ± 1.33) (t = 615.60, 608.40, 524.70, 581.90, P < 0.01; t = 528.10, 520.90, 437.20, 494.40, P < 0.01). Conclusion AgB can up-regulate the expression of Arg-1 in macrophages and polarize it towards M2 type, which may be an important regulatory molecule in the host-parasite immune responses, involving in the immune regulation of macrophages.

Key words: Echinococcus granulosus, Secreted antigen, Antigen B, Polarization of macrophages

中图分类号:

R383.33

焦红杰, 齐文静, 郭刚, 包建玲, 吴川川, 宋传龙, 李军, 张文宝, 严媚. 细粒棘球蚴抗原B对小鼠巨噬细胞RAW264.7的极化作用[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(1): 23-28.

JIAO Hongjie, QI Wenjing, GUO Gang, BAO Jianling, WU Chuanchuan, SONG Chuanlong, LI Jun, ZHANG Wenbao, YAN Mei. Polarization effect of Echinococcus granulosus antigen B on the mouse macrophage RAW264.7[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(1): 23-28.

图/表 2

参考文献 19

[1]	Gordon S, Taylor PR. Monocyte and macrophage heterogeneity[J]. Nat Rev Immunol, 2005, 5(12): 953-964. doi: 10.1038/nri1733 pmid: 16322748
[2]	Li L, Zhuo J, Zheng L, et al. Effects of different induced polarization methods on the proliferation, apoptosis and phagocytosis of rat bone marrow-derived macrophages[J]. Chin J Tissue Eng Res, 2021, 25(25): 4032-4037. (in Chinese)
	(李莉, 卓瑾, 郑玲, 等. 不同诱导极化方式对大鼠骨髓来源巨噬细胞增殖、凋亡及吞噬能力的影响[J]. 中国组织工程研究, 2021, 25(25): 4032-4037.)
[3]	Zheng X, Wang HY. M2 macrophage polarization and the related diseases[J]. Chin Bull Life Sci, 2017, 29(9): 883-890. (in Chinese)
	(郑新, 王红艳. M2型巨噬细胞极化及相关疾病的研究进展[J]. 生命科学, 2017, 29(9): 883-890.)
[4]	Zhao C, Mirando AC, Sové RJ, et al. A mechanistic integrative computational model of macrophage polarization: implications in human pathophysiology[J]. PLoS Comput Biol, 2019, 15(11): e1007468. doi: 10.1371/journal.pcbi.1007468
[5]	Jia R, Hui Y, Yan SG, et al. Research progress on relationship between macrophage M1/M2 polarization and immune inflammatory diseases[J]. Chin J Immunol, 2021, 37(22): 2791-2797. (in Chinese)
	(贾瑞, 惠毅, 闫曙光, 等. 巨噬细胞M1/M2型极化与免疫炎症性疾病关系的研究进展[J]. 中国免疫学杂志, 2021, 37(22): 2791-2797.)
[6]	Mamilos A, Winter L, Schmitt VH, et al. Macrophages: from simple phagocyte to an integrative regulatory cell for inflammation and tissue regeneration: a review of the literature[J]. Cells, 2023, 12(2): 276. doi: 10.3390/cells12020276
[7]	Cai J, Huang L, Wang LJ, et al. The role of macrophage polarization in parasitic infections: a review[J]. Chin J Schisto Control, 2020, 32(4): 432-435.
	(蔡娟, 黄琳, 王灵军, 等. 巨噬细胞极化在寄生虫感染中的作用研究进展[J]. 中国血吸虫病防治杂志, 2020, 32(4): 432-435.)
[8]	Hidalgo C, Stoore C, Baquedano MS, et al. Response patterns in adventitial layer of Echinococcus granulosus sensu stricto cysts from naturally infected cattle and sheep[J]. Vet Res, 2021, 52(1): 66. doi: 10.1186/s13567-021-00936-8
[9]	Silva-Álvarez V, Ramos AL, Folle AM, et al. Antigen B from Echinococcus granulosus is a novel ligand for C-reactive protein[J]. Parasite Immunol, 2018, 40(9): e12575. doi: 10.1111/pim.2018.40.issue-9
[10]	Da Silva ED, Cancela M, Monteiro KM, et al. Antigen B from Echinococcus granulosus enters mammalian cells by endocytic pathways[J]. PLoS Negl Trop Dis, 2018, 12(5): e0006473. doi: 10.1371/journal.pntd.0006473
[11]	Bao JL. Immune regulation in inflammatory bowel disease and intestinal microflora by Echinococcus granulosus infection[D]. Urumqi: Xinjiang Medical University, 2019: 37-54. (in Chinese)
	(包建玲. 细粒棘球蚴感染对炎症性肠病的免疫调节与肠道菌群影响[D]. 乌鲁木齐: 新疆医科大学, 2019: 37-54.)
[12]	Wang JH, Wang N, Hu DD, et al. Genetic diversity of Echinococcus granulosus in southwest China determined by the mitochondrial NADH dehydrogenase subunit 2 gene[J]. Sci World J, 2014, 2014: 867839.
[13]	Zheng Q, Zhang JW, Zuo XS, et al. Photobiomodulation promotes neuronal axon regeneration after oxidative stress and induces a change in polarization from M1 to M2 in macrophages via stimulation of CCL2 in neurons: relevance to spinal cord injury[J]. J Mol Neurosci, 2021, 71(6): 1290-1300. doi: 10.1007/s12031-020-01756-9 pmid: 33417168
[14]	Zhang Y, Qi WJ, Jiao HJ, et al. Antigen B secreted by Echinococcus granulosus reduces asthma by rebalancing Th17/Treg[J]. J Pathog Biol, 2021, 16(8): 927-930, 933. (in Chinese)
	(张耀, 齐文静, 焦红杰, 等. 细粒棘球绦虫分泌抗原B调控Th17/Treg抑制过敏性哮喘的研究[J]. 中国病原生物学杂志, 2021, 16(8): 927-930, 933.)
[15]	AHAN Ayifuhan, Haliya, AJI Tuerganaili, et al. Protective effect of Echinococcus granulosus antigen B on low-dose streptozotocin-induced diabetes mellitus in mice[J]. J Med Postgrad, 2014, 27(5): 452-455. (in Chinese)
	(阿依甫汗•阿汗, 哈丽娅, 吐尔干艾力•阿吉, 等. 细粒棘球蚴抗原B对1型糖尿病小鼠的保护作用[J]. 医学研究生学报, 2014, 27(5): 452-455.)
[16]	Lewandowicz-Uszyńska A, Pasternak G, Świerkot J, et al. Primary immunodeficiencies: diseases of children and adults: a review[J]. Adv Exp Med Biol, 2021, 1289: 37-54.
[17]	Kittivisuit S, Vachvanichsanong P, McNeil E, et al. Childhood-onset systemic lupus erythematosus and immune thrombocytopenia: prevalence and risk factors[J]. Pediatr Blood Cancer, 2021, 68(8): e29146.
[18]	Wang H, Li J, Pu HW, et al. Echinococcus granulosus infection reduces airway inflammation of mice likely through enhancing IL-10 and down-regulation of IL-5 and IL-17A[J]. Parasit Vectors, 2014, 7: 522. doi: 10.1186/s13071-014-0522-6 pmid: 25409540
[19]	Głowińska-Olszewska B, Szabłowski M, Panas P, et al. Increasing co-occurrence of additional autoimmune disorders at diabetes type 1 onset among children and adolescents diagnosed in years 2010—2018-single-center study[J]. Front Endocrinol (Lausanne), 2020, 11: 476. doi: 10.3389/fendo.2020.00476

细粒棘球蚴抗原B对小鼠巨噬细胞RAW264.7的极化作用

Polarization effect of Echinococcus granulosus antigen B on the mouse macrophage RAW264.7

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 2

参考文献 19

相关文章 15

编辑推荐

Metrics

本文评价

[1]	叶井明, 何威, 刘慧媛, 鱼潇, 罗波, 刘美辰, 周必英. 猪囊尾蚴排泄分泌抗原TPx对仔猪树突状细胞活化的影响[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 286-293.
[2]	吴晓莹, 胡媛, 曹建平. 细粒棘球绦虫多肽-壳聚糖季铵盐纳米颗粒的制备[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 300-305.
[3]	李奔福, 王正青, 徐倩, 字金荣, 严信留, 彭佳, 李建雄, 蔡璇, 吴方伟, 杨亚明. 云南省细粒棘球绦虫线粒体co1和nd1基因序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 306-311.
[4]	孙叶挺, 江楠, 姜岩岩, 李腾, 蒋小凤, 曹建平, 沈玉娟. 细粒棘球蚴原头节源外泌体体外刺激髓源抑制性细胞极化的研究[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 175-180.
[5]	田梦潇, 臧小燕, 郭刚, 齐文静, 郭宝平, 任远, 李军, 张文宝. 丝氨酸蛋白酶在细粒棘球绦虫中的表达及活性鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(2): 233-239.
[6]	范俊杰, 韩秀敏, Nur Fazleen Binti Idris, 李锴, 谭青青, 曹纹侨, 李想, 廖鹏, 叶彬. 细粒棘球绦虫蛋白激酶A的生物信息学特征及免疫反应性[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(6): 682-687.
[7]	曹胜魁, 张小凡, 魏玉环, 潘佳明, 曹建平, 沈玉娟, 陈家旭. 细粒棘球蚴感染小鼠肝精氨酸酶的表达和功能研究[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 304-309.
[8]	周鸿让, 茅光耀, 王晓玲, 陈木新, 余晴, 王莹, 艾琳, 肖宁. 重组酶介导的多重核酸等温扩增法鉴别细粒棘球绦虫和多房棘球绦虫技术的建立与应用[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(3): 310-316.
[9]	宋宏宇, 梁雨晴, 华瑞其, 史媛, 阳爱国, 郭莉, 袁东波, 谢跃, 杨光友. 细粒棘球绦虫磷酸甘油酸变位酶的表达、定位和诊断价值的初步评价[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 194-201.
[10]	焦玉萌, 夏惠, 王雪梅, 陶志勇, 朱琳, 方强. 刚地弓形虫排泄分泌抗原刺激NK细胞过继转输对小鼠B16F10黑色素瘤生长的抑制作用[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 444-447.
[11]	魏玉环, 胡媛, 曹建平. 细粒棘球绦虫基因多态性的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 481-485.
[12]	付世强, 樊海宁, 王海久, 周瀛, 曹得萍, 李衍飞, 王志鑫, 任利. 绵羊肝脏、肺脏细粒棘球蚴原头节miRNA表达特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(2): 137-143.
[13]	魏玉环, 胡媛, 曹建平. 抗细粒棘球绦虫疫苗的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 97-101.
[14]	于爱萍，尹建海，巩文词，曹胜魁，曹建平，沈玉娟*. 细粒棘球蚴感染小鼠髓源抑制性细胞与Th17细胞比例的变化[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(3): 5-224-230.
[15]	闫帅, 严萍, 莫筱瑾, 徐斌, 张颋, 胡薇, 詹若挺, 叶萍. 细粒棘球绦虫乳酸脱氢酶B细胞表位的预测及鉴定[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 554-558.