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氧化石墨烯氧化状态/横向尺寸对肝脏细胞死亡机制和炎症反应的影响

陈曦 刘佳尚 洪华

陈曦, 刘佳尚, 洪华. 氧化石墨烯氧化状态/横向尺寸对肝脏细胞死亡机制和炎症反应的影响[J]. 华东理工大学学报(自然科学版), 2021, 47(6): 653-666. doi: 10.14135/j.cnki.1006-3080.20210519001
引用本文: 陈曦, 刘佳尚, 洪华. 氧化石墨烯氧化状态/横向尺寸对肝脏细胞死亡机制和炎症反应的影响[J]. 华东理工大学学报(自然科学版), 2021, 47(6): 653-666. doi: 10.14135/j.cnki.1006-3080.20210519001
CHEN Xi, LIU Jiashang, HONG Hua. Effect of Oxidative State and Lateral Size of Graphene Oxide on Cell Death Mechanisms and Pro-Inflammatory Responses in the Liver[J]. Journal of East China University of Science and Technology, 2021, 47(6): 653-666. doi: 10.14135/j.cnki.1006-3080.20210519001
Citation: CHEN Xi, LIU Jiashang, HONG Hua. Effect of Oxidative State and Lateral Size of Graphene Oxide on Cell Death Mechanisms and Pro-Inflammatory Responses in the Liver[J]. Journal of East China University of Science and Technology, 2021, 47(6): 653-666. doi: 10.14135/j.cnki.1006-3080.20210519001

氧化石墨烯氧化状态/横向尺寸对肝脏细胞死亡机制和炎症反应的影响

doi: 10.14135/j.cnki.1006-3080.20210519001
详细信息
    作者简介:

    陈曦:陈 曦(1986-),女,博士,副教授,研究方向为材料生物学。E-mail:chenxi@ecust.edu.cn

  • 中图分类号: Q291

Effect of Oxidative State and Lateral Size of Graphene Oxide on Cell Death Mechanisms and Pro-Inflammatory Responses in the Liver

  • 摘要: 氧化石墨烯(GO)在药物传递、生物传感和生物成像等方面表现出极佳的性能,但在肝脏等器官的生物相容性方面仍存在问题。分别合成了大(约510 nm)、小(约110 nm)两种尺寸的原始氧化态氧化石墨烯(pGO,根据尺寸不同分别命名为pGO-L和pGO-S)和还原态氧化石墨烯(rGO,根据尺寸不同分别命名为rGO-L和rGO-S);探讨了GO对3种肝细胞(枯氏细胞、肝窦内皮细胞、肝细胞)的生物学影响。结果表明,pGO能诱导枯氏细胞膜损伤并引起其坏死(pGO-L>pGO-S),而对肝窦内皮细胞和肝细胞毒性较小;rGO可诱导枯氏细胞和肝窦内皮细胞凋亡(rGO-L>rGO-S),但对肝细胞影响微乎其微。在溶酶体和NLRP3炎症小体水平上进一步研究GO的胞内效应,结果表明,rGO造成枯氏细胞和肝窦内皮细胞的溶酶体损伤并诱导了白细胞介素-1β(IL-1β)的产生,但rGO在肝细胞中和pGO实验组中均未见类似效应。研究表明GO的表面氧化状态和横向尺寸在肝脏细胞中引发了不同的细胞死亡机制和炎症反应。

     

  • 图  1  GO材料理化性质的表征

    Figure  1.  Physicochemical characterizations of GO materials

    图  2  GO在 Kup5、 SK-HEP-1 和 Hepa 1-6 细胞中的毒性实验

    Figure  2.  Cytotoxicity of GO in Kup5, SK-HEP-1 and Hepa 1-6 cells

    * Stands for P<0.05 compared to cell control based on student's T test; & stands for P<0.05 compared pGO-S with pGO-L based on student’s T test; # stands for P<0.05 compared rGO-S with rGO-L based on student’s T test

    图  3  GO诱导3种肝脏细胞死亡的机制

    Figure  3.  Mechanisms of cell death for three types of liver cells induced by GO

    图  4  GO与肝脏细胞的细胞结合实验

    Figure  4.  Cellular association of GO with liver cells

    Cellular association of pGO and rGO nanosheets in (a) Kup5, (b) SK-HEP-1 and (c) Hepa 1-6 cell lines by fluorescence confocal microscopy and flow cytometry. Cells were treated with 50 μg/mL pGO and rGO nanosheets for 16 h, respectively. (d) TEM images of cellular interaction of pGO-L and rGO-L nanosheets in Kup5 cells after 6 h incubation. Two images of the magnification on the right are the frame area in the middle two images, respectively. (e) Cellular association of GO (50 μg/mL). Cells were pre-treated with BLT-1 for 8 h prior to pGO/rGO exposure at a working concentration of 100 nmol/L. Cellular uptake was quantified by side scatter of flow cytometry

    图  5  GO诱导肝脏细胞脂质过氧化实验

    Figure  5.  Lipid peroxidation test of liver cells induced by GO

    Confocal imaging (up) and percentage (down) of GO induced lipid peroxidation in (a) Kup 5, (b) SK-HEP-1 and (c) Hepa 1-6 cells, respectively. * Stands for P<0.05 compared to cell control based on student's T test

    图  6  Kup5细胞在pGO和rGO(50 μg/mL)中处理6 h后溶酶体损伤及组织蛋白酶B释放

    Figure  6.  Lysosomal damage and cathepsin B release in Kup5 cells after treated by pGO and rGO (50 μg/mL) for 6 h, respectively

    图  7  Kup5,SK-HEP-1 和 Hepa 1-6三种肝脏细胞暴露于pGO和rGO 24 h 后 IL-1β的释放

    Figure  7.  IL-1β production induced by pGO and rGO in Kup5, SK-HEP-1, and Hepa 1-6 after 24 h incubation, respectively

    * Stands for P<0.05 compared to cell control based on  student's  T  test;  # Stands for P<0.05  based  on  student ’s T  test

    图  8  GO 诱导Kup5,SK-HEP-1 和 Hepa 1-6 三种肝脏细胞死亡和促炎症反应机理示意图

    Figure  8.  Schematic illustration of GO induced differential cell death mechanisms and pro-inflammatory responses  in Kup5,  SK-HEP-1, and Hepa 1-6 cells

    表  1  GO材料的氧碳质量比和氧化基团质量分数

    Table  1.   Quantification of oxygen-to-carbon mass ratio and the mass fractions of oxidized groups of GO materials

    GOm(O) : m(C)w(C−C)/%w(C−O)/%w(C=O )/%w(O−C=O )/%
    pGO-S1.0 : (2.2 ± 0.1)48.5 ± 2.244.7 ± 1.86.8 ± 1.1
    pGO-L1.0 : (2.1 ± 0.1)53.8 ± 1.139.7 ± 0.86.5 ± 0.4
    rGO-S1.0 : (6.7 ± 0.1)63.0 ± 2.016.7 ± 0.914.9 ± 1.25.4 ± 0.1
    rGO-L1.0 : (7.7 ± 0.1)64.1 ± 0.715.5 ± 0.715.2 ± 0.75.1 ± 0.2
    下载: 导出CSV

    表  2  pGO和rGO纳米片在细胞培养基中的水合粒径和Zeta电位

    Table  2.   Hydrodynamic size and Zeta potential of pGO and rGO nanosheets in different cell culture media

    MediumHydrodynamic size/nmZeta potential/mV
    pGO-SpGO-LrGO-SrGO-LpGO-SpGO-LrGO-SrGO-L
    Kup5258.9 ± 6.6448.4 ± 19.2383.7 ± 52.5685.1 ± 37.0−13.8 ± 1.6−11.9 ± 2.6−12.9 ± 2.5−12.7 ± 1.0
    SK-HEP-1256.4 ± 7.2556.8 ± 6.3352.4 ± 7.3557.9 ± 16.0−14.7 ± 2.3−14.4 ± 1.2−14.9 ± 2.0−14.7 ± 3.6
    Hepa 1-6278.5 ± 5.0448.6 ± 8.1370.0 ± 12.3765.6 ± 20.5−14.4 ± 2.6−14.6 ± 4.2−14.3 ± 2.9−13.7 ± 1.6
    下载: 导出CSV

    表  3  pGO和rGO在溶酶体模拟液(pH 4.5)中6 h后的水合粒径

    Table  3.   Hydrodynamic sizes of pGO and rGO after suspending in lysosomal simulant fluid (pH 4.5) for 6 h, respectively

    GOHydrodynamic size/nmPDI
    pGO-S829.8 ± 80.90.378
    pGO-L1479.8 ± 25.90.375
    rGO-S12215.2 ± 896.40.472
    rGO-L16214.4 ± 8099.80.476
    下载: 导出CSV
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  • 收稿日期:  2021-05-19
  • 网络出版日期:  2021-06-29
  • 刊出日期:  2021-12-30

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