猪囊尾蚴排泄分泌抗原TPx对仔猪树突状细胞活化的影响

doi:10.12140/j.issn.1000-7423.2023.03.004

中国寄生虫学与寄生虫病杂志 ›› 2023, Vol. 41 ›› Issue (3): 286-293.doi: 10.12140/j.issn.1000-7423.2023.03.004

猪囊尾蚴排泄分泌抗原TPx对仔猪树突状细胞活化的影响

叶井明(), 何威, 刘慧媛, 鱼潇, 罗波, 刘美辰, 周必英^*()

遵义医科大学寄生虫学教研室，贵州遵义 563000

收稿日期:2022-09-06 修回日期:2023-12-29 出版日期:2023-06-30 发布日期:2023-06-20
通讯作者: *周必英（1974-），女，博士，教授，从事寄生虫感染与免疫研究。E-mail: zbyz101@126.com
作者简介:叶井明（2002-），男，本科生，从事寄生虫感染与免疫研究。E-mail：2929201784@qq.com
基金资助:
国家自然科学基金(81960378);贵州省科技厅基础研究计划（黔科合基础-ZK[2023]一般516）;贵州省大学生创新创业训练项目(S202210661169);遵义市校联合基金（遵市科合HZ字[2021]278号）;遵义医科大学学术新苗培养及创新探索专项项目（黔科合平台人才[2020]-004）;遵义医科大学研究生教育创新计划项目(ZYK70);遵义医科大学大学生创新创业训练计划项目(ZYDC2021143)

Effect of excretory-secretory antigen TPx of Cysticercus cellulosae on activation of dendritic cells in piglets

YE Jingming(), HE Wei, LIU Huiyuan, YU Xiao, LUO Bo, LIU Meichen, ZHOU Biying^*()

Department of Parasitology, Zunyi Medical University, Zunyi 563000, Guizhou, China

Received:2022-09-06 Revised:2023-12-29 Online:2023-06-30 Published:2023-06-20
Contact: *E-mail: zbyz101@126.com
Supported by:
National Natural Science Foundation of China(81960378);Basic Research Program of the Department of Science and Technology of Guizhou Province (Qiankehe Foundation-ZK [2023] General 516);Undergraduate Innovation and Entrepreneurship Training Program of Guizhou Province(S202210661169);Zunyi City School Joint Fund ([2021]278);Special Project of Cultivating New Academic Seedlings and Innovative Exploration of Zunyi Medical University (Qian Sci Con Platform Talent[2020]-004);Project of Zunyi Medical University Graduate Education Innovation(ZYK70);Undergraduate Innovation and Entrepreneurship Training Program of Zunyi Medical University(ZYDC2021143)

摘要/Abstract

摘要：

目的探讨猪囊尾蚴排泄分泌抗原（ESA）硫氧还蛋白过氧化物酶（TPx）对仔猪树突状细胞（DC）活化的影响。方法体外诱导培养健康仔猪髓源DC，培养7 d后，加入终浓度为100 ng/ml的脂多糖（LPS）刺激，继续孵育2 d，分别收集培养未成熟DC（imDC）和成熟DC（mDC），光学显微镜和扫描电镜下观察其培养1~9 d的形态学变化，流式细胞术检测其表面标志物CD1和主要组织相容性复合体Ⅱ（MHC-Ⅱ）的表达情况。另收集培养后7 d的imDC，设阴性对照组、TPx组、排泄分泌抗原（ESA）组、LPS阳性对照组，分别加入RPMI 1640培养基、TPx（50 μg/ml）、ESA（50 μg/ml）、LPS（100 ng/ml）刺激48 h后，流式细胞术检测DC表面标志物MHC-Ⅱ、CD80、CD86的表达情况；ELISA检测DC细胞培养上清中肿瘤坏死因子-α（TNF-α）、白细胞介素6（IL-6）、IL-10、IL-12的分泌水平。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。结果光学显微镜下可见，培养第1 天，imDC呈卵圆形，形态单一；随着培养时间延长，DC体积逐渐增大，出现伪足和刺突等，并从卵圆形变为不规则状。扫描电镜下可见，与imDC相比，mDC形态不规则，大致呈长梭形，从胞体辐射出众多长短不一的突起，呈树枝状分布，为典型的树突状结构。流式细胞术检测结果显示，imDC中表达CD1、MHC-Ⅱ的DC占比分别为（0.113 ± 0.005）%、（0.430 ± 0.016）%，低于mDC的（21.400 ± 0.327）%、（21.333 ± 0.450）%（t = 130.341、92.906，均P < 0.05）。TPx组表达MHC-Ⅱ、CD80、CD86的DC占比分别为（15.300 ± 0.245）%、（22.900 ± 0.374）%、（13.033 ± 0.249）%，均低于LPS阳性对照组的（19.000 ± 0.374）%、（31.600 ± 0.082）%、（21.300 ± 0.245）%（t = 11.53、46.32、43.84，均P < 0.05）和ESA组的（18.365 ± 0.618）%、（40.400 ± 0.356）%、（30.300 ± 0.283）%]（t = 9.55、93.17、91.57，均P < 0.05）；TPx组表达MHC-Ⅱ的DC占比高于阴性对照组的（12.133 ± 0.492）%（t = 9.87，P < 0.05）。ELISA检测结果显示，培养上清中，TPx组DC分泌IL-6水平为15.682 ± 0.660，低于阴性对照组的21.041 ± 0.901（t = 6.51，P < 0.05）；分泌TNF-α、IL-6、IL-10和IL-12水平均低于LPS阳性对照组的169.037 ± 7.823、42.118 ± 1.932、34.730 ± 1.772、52.504 ± 2.431（t = 36.79、32.09、13.09、35.05，均P < 0.05）；分泌IL-12水平低于ESA组的23.854 ± 1.020（t = 6.93，P < 0.05）。结论 TPx通过诱导DC表面MHC-Ⅱ高表达、CD80和CD86低表达，以及降低IL-6的分泌水平来介导免疫耐受。

关键词: 猪囊尾蚴, 排泄分泌抗原, 硫氧还蛋白过氧化物酶, 树突状细胞

Abstract:

Objective This study investigates the effect of thioredoxin peroxidase (TPx) in the excretory-secretory antigen (ESA) of Cysticercus cellulosae on activation of dendritic cell (DC) in piglets. Methods Healthy piglet medulla-derived DC were cultured in vitroo, in which lipopolysaccharide (LPS) was added at a final concentration of 100 ng/ml on 7 d for stimulation for 2 days, and then continuously cultured for 2 more days to collect immature DC (imDC) and mature DC (mDC) separately. The morphological changes of DCs were observed by light microscopy and scanning electron microscopy on 1 to 9 d of culture. The expression of surface markers CD1 and major histocompatibility complex (MHC-Ⅱ) was detected by flow cytometry. The 7 d imDC was used in the assay with the assigned groups of negative control, TPx, ESA and LPS positive control, to which RPMI 1640 medium, TPx (50 μg/ml), ESA (50 μg/ml) and LPS (100 ng/ml) was added, respectively, to stimulate for 48 h for examining the expression of DC surface markers MHC-Ⅱ, CD80 and CD86 using flow cytometry and for detecting secretion levels of tumor necrosis factor-α (TNF-α), interleukin (IL-6), IL-10, IL-12 in DC culture supernatant by ELISA. One-way ANOVA was used for comparison between multiple groups, and LSD-t test was used for two-way comparison between groups. Results Under ligth microscope, imDC were ovoid in shape with single form at the first day of culture; with the extension of culture time, DC increased in size, appeared pseudopods and spines and other features, and changed from ovoid to irregular shape. Scanning electron microscopy showed that compared with imDC, mDC had irregular morphology, roughly long shuttle shape, and numerous protrusions of different lengths radiating from the cytosol, which were distributed in a dendritic pattern, a typical dendritic structure. Flow cytometry showed that the expression of CD1 and MHC-Ⅱ in imDC was (0.113 ± 0.005)% and (0.430 ± 0.016)%, respectively, which was lower than that of mDC (21.400 ± 0.327)% and (21.333 ± 0.450)% (t = 130.341, 92.906, both P < 0.05). The expression levels of MHC-Ⅱ, CD80, and CD86 in the TPx group were (15.300 ± 0.245)%, (22.900 ± 0.374)% and (13.033 ± 0.249)%, respectively, which were lower than those in the LPS positive control group (19.000 ± 0.374)%, (31.600 ± 0.082)%, and (21.300 ± 0.245)% (t = 11.53, 46.32, 43.84, all P < 0.05) and the ESA group (18.365 ± 0.618)%, (40.400 ± 0.356)% and (30.300 ± 0.283)%] (t = 9.55, 93.17, 91.57, all P < 0.05). The MHC-Ⅱ expression level in the TPx group was higher than that of the negative control group (12.133 ± 0.492)% (t = 9.87, P < 0.05). ELISA results showed that IL-6 level in DC of the TPx group was 15.682 ± 0.660, which was ower than that of 21.041 ± 0.901 in the control group (t = 6.51, P < 0.05); TNF-α (35.711 ± 4.196), IL-6, IL-10 (22.216 ± 1.357) and IL-12 (16.799 ± 0.523) were all lower than those of the LPS positive control group 169.037 ± 7.823, 42.118 ± 1.932, 34.730 ± 1.772, 52.504 ± 2.431 (t = 36.79, 32.09, 13.09, 35.05, all P < 0.05); IL-12 level were lower than that of the ESA group at 23.854 ± 1.020 (t = 6.93, P < 0.05). Conclusion TPx mediates immune tolerance by inducing high expression of DC surface molecules MHC-Ⅱ, low expression of CD80 and CD86, and reducing the secretion levels of IL-6.

Key words: Cysticercus cellulosae, Excretory-secretory antigens, Thioredoxin peroxidase, Dendritic cell

中图分类号:

R383.34

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.

YE Jingming, HE Wei, LIU Huiyuan, YU Xiao, LUO Bo, LIU Meichen, ZHOU Biying. Effect of excretory-secretory antigen TPx of Cysticercus cellulosae on activation of dendritic cells in piglets[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(3): 286-293.

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猪囊尾蚴排泄分泌抗原TPx对仔猪树突状细胞活化的影响

Effect of excretory-secretory antigen TPx of Cysticercus cellulosae on activation of dendritic cells in piglets

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