不同来源细菌内毒素光谱特征及其对单核细胞免疫效应的影响

Spectral characteristics of bacterial endotoxin from different sources and their effects on the immune response of monocytes

中文关键词:  内毒素  鲎试验  单核细胞活化反应测定法  免疫效应  Toll样受体

英文关键词:endotoxin  Limulus amebocyte lysate assay  monocyte activation test  immune response  Toll-like receptors

中文摘要:

      目的 比较不同来源细菌内毒素的光谱特征以及对单核细胞的免疫效应差异。方法 采用紫外吸收光谱和三维荧光光谱识别5种内毒素工作标准品中非内毒素成分如赋形剂(聚乙二醇8000和α-乳糖)和生物杂质(蛋白质和核酸), 结合HL-60细胞Toll样受体(Toll-like receptors, TLRs)测试进一步对生物杂质成分进行识别, 通过单核细胞活化反应测定法比较5种内毒素诱导HL-60细胞释放炎症因子白细胞介素-6 (interleukin 6, IL-6)、白细胞介素-1β (interleukin-1β, IL-1β)和肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)情况。结果 内毒素工作标准品紫外吸收光谱中出现与赋形剂相关的吸收峰, 三维荧光光谱中出现与赋形剂和蛋白质相关的荧光峰。HL-60细胞TLRs测试结果表明内毒素中蛋白质污染相较于核酸污染更明显。5种内毒素均引起HL-60细胞IL-6和IL-1β基因相对表达水平以及蛋白质量浓度升高, TNF-α基因相对表达水平及蛋白质量浓度未升高, 结果表明相同或不同菌种来源的内毒素在同一效价条件下的炎症效应差异明显。结论 光谱检测识别内毒素的赋形剂和蛋白质污染, HL-60细胞TLRs测试对生物污染成分进一步确认。内毒素分子结构差异以及非内毒素成分的蛋白质污染, 导致相同或不同菌种来源内毒素诱导炎症因子释放能力产生差异。

英文摘要:

      Objective To compare the differences in spectral characteristics and monocyte immune effects for bacterial endotoxin from different sources. Methods Ultraviolet absorption spectrum and three-dimensional fluorescence spectrum were used to identify excipients (polyethylene glycol 8000 and α-lactose) and biological impurities (protein and nucleic acid) in 5 kinds of control standard endotoxin. Biological contamination was further confirmed by the detection of Toll-like receptors (TLRs) in HL-60 cells. The monocyte activation test was used to compare the release of the inflammatory cytokines interleukin 6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) caused by 5 kinds of endotoxins on HL-60 cells. Results As for control standard endotoxin, absorption peaks related to excipients were observed in ultraviolet absorption spectrum, while fluorescence peaks related to excipients and proteins were shown in three-dimensional fluorescence spectrum. The expression levels of TLRs of HL-60 cells?indicated that protein contamination was more significant than nucleic acid contamination. All endotoxins induced improvement in cellular IL-6 and IL-1β gene relative expression levels and their protein mass concentrations in HL-60 cells. However, the TNF-α gene relative expression levels and its protein concentrations were not elevated. Endotoxins from the same or different bacterial strains had significant differences in their ability to induce inflammation under the same endotoxin activities. Conclusion Excipients and protein contamination of endotoxins are identified by spectral detection, while proteins and nucleic acids in biological impurity are confirmed by the TLRs assay of HL-60 cells. Molecular structure of endotoxins, as well as protein contamination influence the ability of endotoxins to induce the release of the inflammatory cytokines.