目的 观察体外培养的星形胶质细胞在炎性刺激下的激活及功能变化。 方法 体外培养 C57BL/6 小鼠皮层的星形胶质细胞，应用免疫细胞化学和Western blot等方法，测定炎性刺激下各组细胞形态学及促炎因子（iNOS、IL-6、TNF-α）和抗炎因子（ARG1、IL-10、TGF-β1）表达水平的变化情况。 结果 炎性刺激后，免疫荧光细胞染色结果显示，M1（LPS+IFN-γ）组细胞被异常激活，胞体出现肿胀变大、扭曲等形态改变，且GFAP阳性细胞数量明显增多（P<0.001 vs. control））。CCK-8细胞活力检测结果显示，LPS组、IFN-γ组及M1组的吸光度均升高，其中M1组显著升高（P<0.001 vs. control）。Western blot结果表明，M1组促炎因子（iNOS、IL-6和TNF-α）表达均明显升高（P<0.001 vs. control）；各组抗炎因子（ARG1、IL-10 和TGF-β1）的表达也显著升高（P<0.01 or 0.001 vs. control）。 结论 体外炎性刺激异常激活星形胶质细胞，形态和功能显著变化，且A1样星形胶质细胞的炎性因子表达水平升高。
关键词：
星形胶质细胞; ; ; 脂多糖; ; ; 炎性刺激; ; ; 神经炎症
Objective To observe the activation and functional changes of astrocytes cultured in vitro under inflammatory stimulation. Methods Astrocytes from the cortex of C57BL/6 mice were isolated and cultured in vitro, the changes of astrocyte morphology were observed by immunocytochemistry and the expression levels of proinflammatory factors (iNOS, IL-6, TNF-α) and anti-inflammatory factors (ARG1, IL-10, TGF-β1) were determined by Western blot protein analysis. Results After inflammatory stimulation, immunofluorescence cell staining showed that astrocytes in M1（LPS+IFN-γ） group were abnormally activated, cell body swelling, enlargement, distortion and other morphological changes were also obvious, and the number of GFAP positive astrocytes in M1 group were evidently increased (P<0.001 vs. control). CCK-8 test showed that compared with the control group, the absorbance values of LPS group, IFN-γ group and M1 group were increased, especially the absorbance values of M1 group was significantly increased (P<0.001 vs. control). Western blot results showed that the expression levels of iNOS, IL-6 and TNF-α in M1 group were markedly increased after LPS and IFN-γ combined stimulation (P<0.001 vs. control). The expression levels of anti- inflammatory factors (ARG1, IL-10 and TGF-β1) in each group were also significantly upregulated (P<0.01 or 0.001 vs. control). Conclusions Under inflammatory stimulation, the cultured-astrocytes in vitro can be abnormally activated, its morphological and functional changes are distinct, and the expression level of A1-like inflammatory factors in astrocytes are increased.
[1] Grigoriadis N, van Pesch V, Paradig MSG. A basic overview of multiple sclerosis immunopathology[J]. Eur J Neurol, 2015, 22(Suppl2): 3-13. DOI: 10.1111/ene.12798.
[2] Sofroniew MV. Multiple roles for astrocytes as effectors of cytokines and inflammatory mediators[J]. Neuroscientist, 2014, 20(2): 160-172. DOI: 10.1177/1073858413504466.
[3] Niranjan R. The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson’s disease: focus on astrocytes[J]. Mol Neurobiol, 2014, 49(1): 28-38. DOI: 10.1007/s12035-013-8483-x.
[4] Koob AO. Astrogenesis versus astrogliosis[J]. Neural Regen Res, 2017, 12(2): 203-204. DOI: 10.4103/1673-5374.200798.
[5] Masuda T, Sankowski R, Prinz M, et al. Microglia heterogeneity in the single-cell era[J]. Cell Reports, 2020, 30(5): 1271-1281. DOI: 10.1016/j.celrep.2020.01.010.
[6] Glass CK, Saijo K, Gage FH, et al. Mechanisms underlying inflammation in neurodegeneration[J]. Cell, 2010, 140(6): 918-934. DOI: 10.1016/j.cell.2010.02.016.
[7] Morita M, Ikeshima-Kataoka H, Noda M, et al. Metabolic Plasticity of Astrocytes and Aging of the Brain[J]. Int J Mol Sci, 2019, 20(4): 941. DOI: 10.3390/ijms20040941.
[8] Kery R, Chen APF, Kirschen GW. Genetic targeting of astrocytes to combat neurodegenerative disease[J]. Neural Regen Res, 2020, 15(2): 199-211. DOI: 10.4103/1673-5374.265541.
[9] Ajmone-Cat MA, Mancini M, Minghetti L, et al. Microglial polarization and plasticity: evidence from organotypic hippocampal slice cultures[J]. Glia. 2013, 61(10): 1698-1711. DOI: 10.1002/glia.22550.
[10]Zhang K, Zheng J, Wang J, et al. Polarized macrophages have distinct roles in the differentiation and migration of embryonic spinal-cord-derived neural stem cells after grafting to injured sites of spinal cord[J]. Mol Ther. 2015, 23(6):1077-1091. DOI: 10.1038/mt.2015.46.
[11]Sochocka M, Diniz BS, Leszek J. Inflammatory response in the CNS: friend or foe[J]? Mol. Neurobiol, 2017, 54(10): 8071-8089. DOI: 10.1007/s12035-016-0297-1.
[12]Sofroniew MV. Astrocyte barriers to neurotoxic inflammation[J]. Nat Rev Neurosci. 2015, 16(5):249-63.DOI: 10.1038/nrn3898.
[13]Cekanaviciute E, Dietrich HK, Wai KM, et al. Astrocytic TGF-β signaling limits inflammation and reduces neuronal damage during central nervous system Toxoplasma infection[J]. J Immunol. 2014,193(1):139-49.DOI: 10.4049/jimmunol. 1303284.
[14]Cynthia JM Kane, Paul D Drew. Neuroinflammatory contribution of microglia and astrocytes in fetal alcohol spectrum disorders[J]. J Neurosci Res, 2021,99(8):1973-1985. DOI: 10.1002/jnr.24735.
[15]Sidoryk-Wegrzynowicz M, Strużyńska L. Dysfunctional glia: contributors to neurodegenerative disorders[J]. Neural Regen Res, 2021, 16(2): 218-222. DOI: 10.4103/1673-5374.290877.
[16]Li KY, Li JT, Qin S, et al. Reactive Astrocytes in Neurodegenerative Diseases[J]. Aging and Disease, 2019, 10(3): 664-675. DOI: 10.14336/AD.2018.0720.
京ICP备08002354号-3
