大片形吸虫外泌体对水牛外周血单核巨噬细胞极化的影响

Regulatory effects of exosomes derived from Fasciola gigantica adult worms on the polarization of peripheral blood mononuclear macrophages in buffalo

为探讨大片形吸虫外泌体(FgExo)在宿主免疫调节方面的作用,本研究采用聚合物沉淀法和超速离心法从大片形吸虫分泌排泄产物(FgESP)中分离得到FgExo;采用荧光定量PCR(qPCR)法检测不同浓度的FgESP或FgExo作用于水牛外周血单核巨噬细胞(Mφ)后γ-干扰素(IFN-γ)、白介素-10(IL-10)的转录水平;根据qPCR结果选择50μg/mL FgExo(+5μg/mL脂多糖)、200μg/mL FgESP(+5μg/mL脂多糖)作为试验组,以PBS为阴性对照组、脂多糖为阳性对照组,分别作用于Mφ后采用qPCR检测Mφ分型相关基因诱导型一氧化氮合酶(iNOS)、CD86、精氨酸酶2(Arg-2)、CD206的转录水平;ELISA检测白介素-1β(IL-1β)、转化生长因子-β1(TGF-β1)分泌水平。结果显示:FgExo和FgESP可显著上调MφIL-10 mRNA的转录水平,显著抑制IFN-γmRNA的转录水平;显著上调M2型Mφ标记物CD206及相关因子Arg-2、TGF-β1的表达,显著抑制M1型Mφ标记物CD86及相关因子iNOS、IL-1β的表达。本研究结果表明,FgExo和FgESP均能够使水牛外周血单核巨噬细胞向M2型极化,利于大片形吸虫在水牛体内生存。

In order to explore the role of Fasciola gigantica exosomes(FgExo)in the immune regulation in the host, exosomes were isolated from the excretory-secretory products of Fasciola gigantica(FgESP) by polymer precipitation separation and ultracentrifugation. Then, qPCR was used to analyze IFN-γ and IL-10 at the mRNA transcript level in the peripheral blood mononuclear macrophages(Mφ) in buffalos stimulated with different concentrations of FgExo and ESP. Based on the qPCR results, 50 μg/mL FgExo(+ 5 μg/mL LPS) and 200 μg/mL FgESP(+ 5 μg/mL LPS) were used as the experimental group, and PBS was used as the negative control and LPS as the positive control to treat Mφ respectively. Next, the transcription levels of Mφ typing related genes iNOS, CD86, Arg-2 and CD206 were detected by qPCR, and the secretion levels of cytokines IL-1β and TGF-β1 were detected by ELISA. The results showed that the exosome and ESP promoted the transcription of IL-10 and inhibited the transcription of IFN-γ, that they significantly upregulated the expression of M2 Mφ marker CD206 and the related factors Arg-2 and TGF-β1, but significantly inhibited the expression of M1 Mφ marker CD86 and the related factors iNOS and IL-1 β. These results indicated that both the Fasciola gigantica exosomes and ESP were able to polarize the peripheral blood derived monocyte macrophages of buffalos toward the M2 type, and were helpful for the survival of Fasciola gigantica in hosts.