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

作　　者：叶井明 何威 刘慧媛 鱼潇 罗波 刘美辰 周必英 YE Jingming;HE Wei;LIU Huiyuan;YU Xiao;LUO Bo;LIU Meichen;ZHOU Biying(Department of Parasitology,Zunyi Medical University,Zunyi 563000,Guizhou,China)

机构地区：[1]遵义医科大学寄生虫学教研室,贵州遵义563000

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第3期286-293,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：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)。

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(50μg/ml)、ESA(50μg/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Objective This study investigates the effect of thioredoxin peroxidase(TPx)in the excretory⁃se⁃cretory 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 concentra⁃tion of 100 ng/ml on 7 d for stimulation for 2 days,and then continuously cultured for 2 more days to collect imma⁃ture DC(imDC)and mature DC(mDC)separately.The morphological changes of DCs were observed by light micros⁃copy and scanning electron microscopy on 1 to 9 d of culture.The expression of surface markers CD1 and major histo⁃compatibility complex(MHC⁃Ⅱ)was detected by flow cytometry.The 7 d imDC was used in the assay with the as⁃signed groups of negative control,TPx,ESA and LPS positive control,to which RPMI 1640 medium,TPx(50μg/ml),ESA(50μg/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 ir⁃regular morphology,roughly long shuttle shape,and numerous protrusions of different lengths radiating from the cyto⁃sol,which were distributed in a dendritic pattern,a typical dendritic structure.Flow cytometry showed that the ex⁃pression of CD1 and MHC⁃Ⅱin 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

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 

分 类 号：R383.34[医药卫生—医学寄生虫学]