李清如, 王祎星, 祁宇泽, 权会会, 周辉. 氧化石墨烯片层对小胶质细胞神经免疫效应的调控[J]. 环境与职业医学, 2020, 37(5): 461-467. DOI: 10.13213/j.cnki.jeom.2020.19735
引用本文: 李清如, 王祎星, 祁宇泽, 权会会, 周辉. 氧化石墨烯片层对小胶质细胞神经免疫效应的调控[J]. 环境与职业医学, 2020, 37(5): 461-467. DOI: 10.13213/j.cnki.jeom.2020.19735
LI Qingru, WANG Yi-xing, QI Yu-ze, QUAN Hui-hui, ZHOU Hui. Effects of graphene oxide slice on neuro-immunity modulation of microglia cells[J]. Journal of Environmental and Occupational Medicine, 2020, 37(5): 461-467. DOI: 10.13213/j.cnki.jeom.2020.19735
Citation: LI Qingru, WANG Yi-xing, QI Yu-ze, QUAN Hui-hui, ZHOU Hui. Effects of graphene oxide slice on neuro-immunity modulation of microglia cells[J]. Journal of Environmental and Occupational Medicine, 2020, 37(5): 461-467. DOI: 10.13213/j.cnki.jeom.2020.19735

氧化石墨烯片层对小胶质细胞神经免疫效应的调控

Effects of graphene oxide slice on neuro-immunity modulation of microglia cells

  • 摘要: 背景

    氧化石墨烯(GO)广泛应用于神经生物学领域。近年来研究表明,GO能够参与神经系统的调节过程,但关于其神经免疫效应调控具体机制尚无相关报道。

    目的

    探究GO在脂多糖(LPS)和白细胞介素4(IL-4)诱导小胶质细胞M1经典活化表型与M2选择活化表型转化中的神经调控作用。

    方法

    在GO基底培养基和对照培养基上对BV2细胞进行培养,分别加入20 μg·L-1 LPS或IL-4诱导BV2细胞表型转化,分组为空白对照组、GO对照组、空白-LPS组、空白-IL-4组、GO-LPS组和GO-IL-4组。比较24 h后培养上清中的细胞数量。利用实时荧光定量PCR(RT-qPCR)和流式细胞术分别检测BV2细胞表面M1表型标志物如肿瘤坏死因子αTNF-α)、诱导型一氧化氮合酶(iNOS)、细胞因子CD86以及M2表型标志物如重组人精氨酸酶1(Arg1)、白细胞介素-10(IL-10)、细胞因子CD206表达量的变化,同时使用分光光度法检测培养上清中一氧化氮(NO)的含量。

    结果

    与空白对照组相比,GO处理后的BV2细胞细胞计数降低(218.13±8.77和175.63±8.24,P < 0.01)。相比于空白-LPS组,GO-LPS组BV2细胞上M1表型标志物TNF-α、iNOS的mRNA相对表达量(8.16±0.26和5.35±0.42;45.64±2.03和30.39±1.84)以及CD86阳性细胞率(14.65±0.85)%和(10.29±0.46)%降低,并且培养上清中NO的含量(19.62±1.07)μmol·L-1和(8.37±1.25)μmol·L-1降低,M2表型标志物IL-10的mRNA相对表达量升高(1.63±0.09和2.09±0.15)(P < 0.05)。与空白-IL-4组相比,GO-IL-4组M1表型标志物iNOS的mRNA相对表达量增加(0.41±0.03和1.05±0.07),M2表型标志物Arg1以及IL-10的mRNA相对表达量下降(201.63±12.31和4.74±0.38;2.63±0.14和0.71±0.13),CD206的阳性细胞率和平均荧光强度下降(17.01±1.03)%和(3.66±0.41)%;8893.2±347.62和1 299.9±159.67(P < 0.01)。

    结论

    GO抑制BV2小胶质细胞生长,并对LPS和IL-4诱导下的神经免疫效应所引起的BV2小胶质细胞向M1和M2表型的转化起到抑制作用。

     

    Abstract: Background

    Graphene oxide (GO) is widely applied to neuroscience. Recent studies have shown that GO can regulate the nervous system, but there are limited reports about the mechanism underlying its neuro-immunity modulation.

    Objective

    This experiment is designed to investigate the regulation of GO towards M1/M2 polarization of microglia induced by lipopolysaccharide (LPS) and interleukin-4 (IL-4).

    Methods

    BV2 cells cultured on GO substrate and control medium were added with 20μg·L-1 LPS and IL-4 respectively to induce M1/M2 phenotype of BV2 cells, and were divided into blank control group, GO control group, LPS control group, IL-4 control group, GO-LPS group, and GO-IL-4 group. After 24 h of incubation, the number of cells was counted. The expression levels of M1 markerstumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS), and cytokine CD86 and M2 markersrecombinant human arginase-1 (Arg1), interleukin-10 (IL-10), and cytokine CD206) were detected by real-time fluorescence quantitative PCR (RT-qPCR) and flow cytometry, and the NO concentration in supernatant was measured by spectrophotometry.

    Results

    After the designed GO treatment, the number of BV2 cells was decreased (218.13±8.77 and 175.63±8.24) compared with the blank control group (P < 0.05). The mRNA expression levels of M1 markers TNF-α (8.16±0.26 and 5.35±0.42), iNOS (45.64±2.03 and 30.39±1.84), CD86 positive rate(14.65±0.85)% and (10.29±0.46)%, and NO production(19.62±1.07) μmol·L-1 and (8.37±1.25) μmol·L-1 were all decreased in the GO-LPS group, as well as the mRNA expression levels of M2 marker IL-10 (1.63±0.09 and 2.09±0.15) increased, compared with the LPS control group (P < 0.05). Under the IL-4 inducement, the mRNA expression level of M1 marker iNOS was upregulated (0.41±0.03 and 1.05±0.07), the levels of Arg1 (201.63±12.31 and 4.74±0.38) and IL-10 (2.63±0.14 and 0.71±0.13) were downregulated, and the positive rate(17.01±1.03)% and (3.66±0.41)% and average fluorescence intensity (8 893.2±347.62 and 1 299.9±159.67) in the GO-IL-4 group (P < 0.01).

    Conclusion

    GO inhibits the growth of BV2 cells and regulates the activation of microglia to resist the effects of LPS/IL-4 induced microglial polarization.

     

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