绵羊痒螨巨噬细胞迁移抑制因子重组蛋白对兔外周血单个核细胞Th1/Th2型和Th17/Treg型特征因子表达的影响

doi:10.11843/j.issn.0366-6964.2020.03.019

摘要/Abstract

摘要： 为了初步探讨绵羊痒螨巨噬细胞迁移抑制因子（PoMIF）对健康新西兰兔外周血单个核细胞（PBMC）中Th1/Th2和Th17/Treg细胞平衡的变化。采用RT-PCR从绵羊痒螨总RNA中扩增得到MIF全长基因，经原核表达、纯化重组PoMIF（rPoMIF）蛋白，并分析其氧化还原酶和互变异构酶活性。筛选与健康新西兰兔PBMC孵育的最佳rPoMIF浓度，且用最佳浓度rPoMIF（0.2 μg·mL^-1）与PBMC共同孵育0、1、6、12、24、36 h后收集细胞，用荧光定量PCR检测其Th1/Th2/Th17/Treg细胞相对应的特征性转录因子T-bet/GATA-3/RORc/Foxp3和特征性细胞因子IFN-γ/IL-4/IL-17A/IL-10 mRNA表达变化。结果表明：PoMIF全长363 bp，rPoMIF大小为32 ku（含pET32a标签蛋白19 ku），且具有互变异构酶活性；rPoMIF刺激后PBMC中Th1细胞的T-bet和IFN-γ先下降后上升，Th2细胞的GATA-3和IL-4均呈下降趋势，且T-bet/GATA-3和IFN-γ/IL-4比值在12、24和36 h均升高；Th17细胞的RORc和IL-17A在各时间点下降，而Treg细胞的Foxp3和IL-10在各时间点升高，且RORc/Foxp3和IL-17A/IL-10比值在各时间点均变低。rPoMIF可造成家兔外周血单个核细胞的Th1/Th2和Th17/Treg平衡分别向Th1和Treg偏移。

关键词: 绵羊痒螨, 兔, 巨噬细胞迁移抑制因子, 外周血单个核细胞, Th1/Th2平衡, Th17/Treg平衡

Abstract: To explore the effects of macrophage migration inhibitory factor of Psoroptes ovis (PoMIF) on Th1/Th2 and Th17/Treg balance of rabbit peripheral blood mononuclear cells (PBMC) in vitro, the PoMIF gene was amplified from the extracted total RNA from P. ovis mites by the reverse transcription-polymerase chain reaction (RT-PCR), and the recombinant protein rPoMIF was expressed with prokaryotic expression system, then the oxidoreductase and tauromerase activities of rPoMIF was evaluated. PBMCs isolated from healthy New Zealand rabbits were cultured with different concentrations of rPoMIF, and the optimum concentration of rPoMIF was screened. Finally, the expression levels of Th1, Th2, Th17 and Treg-specific transcription factors of T-bet, GATA-3, RORc, Foxp3 and specific cytokines of IFN-γ, IL-4, IL-17A and IL-10 in PBMC incubated with the optimal concentration of rPoMIF for 0, 1, 6, 12, 24 and 36 h were detected by real time fluorescence quantitative PCR (qPCR). The results showed that the full-length of PoMIF was 363 bp, containing 357 bp open reading frame; rPoMIF was mainly in the supernatant of Escherichia coli (E. coli) lysate, with a molecular size of 32 kD (including an extra 19 kD for the attached His-tag fusion peptide); and it has tautomerism activity; the mRNA levels of Th1-specific T-bet and IFN-γ decreased firstly and then increased after treatment, while Th2-specific GATA-3 and IL-4 decreased at all tested time points, and the ratio of T-bet/GATA-3 and IFN-γ/IL-4 increased at 12, 24 and 36 h; the mRNA levels of Th17-specific RORc and IL-17A decreased at all tested time points, while Foxp3 and IL-10 from Treg cells increased at all tested time points, and the ratio of RORc/Foxp3 and IL-17A/IL-10 decreased at all tested time points. In conclusion, rPoMIF can cause the shifts of Th1/Th2 and Th17/Treg towards Th1 and Treg in PBMC of rabbits.

Key words: Psoroptes ovis, rabbit, macrophage migration inhibitory factor, peripheral blood mononuclear cells, Th1/Th2 cell balance, Th17/Treg cell balance

中图分类号:

S852.746

郑友乐, 陈宇航, 谢跃, 杨光友, 何冉, 古小彬. 绵羊痒螨巨噬细胞迁移抑制因子重组蛋白对兔外周血单个核细胞Th1/Th2型和Th17/Treg型特征因子表达的影响[J]. 畜牧兽医学报, 2020, 51(3): 594-601.

ZHENG Youle, CHEN Yuhang, XIE Yue, YANG Guangyou, HE Ran, GU Xiaobin. The Effects of Recombinant Macrophage Migration Inhibitory Factor of Psoroptes ovis on the Characteristic Molecules of Th1/Th2 and Th17/Treg Type Immune Responses in Rabbits Peripheral Blood Mononuclear Cells in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 594-601.

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