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单增李斯特菌膜囊泡的提取及其表征与免疫效应分析

Extraction, Characterization and Immune Effect Analysis of Listeria Monocytogenes Membrane Vesicles

  • 摘要:
      目的  建立提取单增李斯特菌膜囊泡(Listeria monocytogenes membrane vesicles, LM-MVs)的方法,对LM-MVs表征及诱导固有免疫应答的能力进行分析,为LM-MVs作为疫苗载体和药物递送平台的研究奠定基础。
      方法  通过培养→离心→过滤→超滤浓缩→超速离心的方法提取单增李斯特菌分泌的膜囊泡,通过透射电子显微镜观察其形态特征,通过动态光散射分析测量其粒径分布,采用SDS-PAGE和Western blot分析其蛋白表达情况,采用CCK-8细胞增殖与毒性实验分析其对固有免疫细胞增殖能力的影响,采用实时荧光定量PCR对其刺激的固有免疫应答进行分析。
      结果  成功建立提取LM-MVs的方法。透射电子显微镜下,LM-MVs呈现近圆形的膜状结构,动态光散射分析显示其大小为65~190 nm。SDS-PAGE和Western blot结果表明LM-MVs含有单增李斯特菌溶血素等蛋白。CCK-8细胞增殖与毒性实验中,10、20和50 μg/mL的LM-MVs干预小鼠树突状细胞(DC 2.4)24 h后的增殖指数均高于未干预的DC 2.4细胞(P<0.05);0.1、1、10、20和50 μg/mL的LM-MVs干预小鼠巨噬细胞(Raw 264.7)24 h后的增殖指数均高于未干预的Raw 264.7细胞(P<0.01)。实时荧光定量PCR结果显示,50 μg/mL的LM-MVs干预Raw 264.7细胞后肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、白细胞介素(interleukin, IL)-1β、IL-6和IL-10的表达量均高于未干预对照(P<0.05)。
      结论  本研究建立的方法能够提取出单增李斯特菌分泌的膜囊泡,提取的LM-MVs直径为65~190 nm近圆形膜状结构,能分泌多种蛋白组分,能刺激固有免疫应答。

     

    Abstract:
      Objective  To establish a method for extracting Listeria monocytogenes membrane vesicles (LM-MVs) and to analyze the characteristics of LM-MVs and their ability to induce innate immune effect in vitro so as to lay the foundation for research into using LM-MVs as vaccine carrier and drug delivery platform.
      Methods  The membrane vesicles secreted by Listeria monocytogenes were extracted through a continuous process, including culturing, centrifugation, filtration, ultrafiltration concentration and ultracentrifugation. The morphological characteristics of LM-MVs were observed with transmission electron microscope, and particle size distribution were measured by dynamic light scattering analysis. SDS-PAGE and Western blot were used to analyze the protein composition of LM-MVs. CCK-8 cell proliferation and toxicity determination experiments were done to analyze their effect on the proliferation of innate immune cells, and qPCR was used to analyze their ability to induce innate immune responses.
      Results  A method for extracting LM-MVs was successfully established. Under the transmission electron microscope, LM-MVs presented a nearly circular film-like structure, and dynamic light scattering analysis showed that their sizes were between 65 and 190 nm. SDS-PAGE and Western blot showed that LM-MVs contained proteins, including listeriolysin O (LLO). CCK-8 cell proliferation and toxicity experiment showed that after intervention with 10, 20 and 50 μg/mL of LM-MVs for 24 hours, the proliferation rate of DC 2.4 mouse dendritic cell line was higher than that of non-interventional DC 2.4 cells (P<0.05); after intervention with 0.1, 1, 10, 20 and 50 μg/mL of LM-MVs for 24 hours, the proliferation rate of RAW 264.7 cells was higher than that of non-interventional RAW 264.7 cells (P<0.01). The results of qPCR showed that, after intervention with 50 μg/mL of LM-MVs, the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-10 in RAW 264.7 cells were higher than those of non-intervention control cells (P<0.05).
      Conclusions  The method established in the study can be used to extract LM-MVs. The extracted LM-MVs have a diameter of 65-190 nm and a nearly circular membrane-like structure. They can secrete a variety of protein components and stimulate innate immune responses.

     

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