新视角重探小鼠胶质细胞体外培养技术与炎症模型
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摘要
目的通过比较新材料与传统材料在胶质细胞培养中的优劣及特点,优化小鼠胶质细胞炎症模型。方法利用q PCR、流式细胞术、免疫荧光等手段,表征胶质细胞组成比例与细胞纯度。在LPS刺激下,从mRNA与蛋白水平,评估不同包被材料对细胞炎症反应的影响。结果 LPS(100μg·L~(-1))刺激48 h,混合胶质细胞炎症因子表达量明显高于小胶质细胞,如PDL包被时,混合胶质细胞TNF-α表达量为(903. 8±322. 2) ng·L~(-1),明显高于小胶质细胞(565. 4±159. 8) ng·L~(-1); Matrigel包被时,混合胶质细胞表层的小胶质细胞状态更佳。而小胶质细胞在未包被时,对LPS更为敏感,TNF-α水平为(6861. 4±1606. 6) ng·L~(-1)。结论 Matrigel包被培养的混合胶质细胞,可最大程度模拟体内环境,为建立胶质细胞炎症模型的最佳条件;评估炎症模型应以蛋白水平检测结果为主要指标。
Aim To revisit the long-established murine glial culture-base inflammation model,in order to explore the possible improvements of current methods. Methods The proportion and purity of glial cells were characterized by q PCR,flow cytometry and immunofluorescence. After stimulation with LPS,their inflammation response was evaluated at both mRNA and protein levels. Results Mixed glial cells were stimulated by LPS( 100μg·L~(-1)),and the expression of inflammatory factors increased more significantly than that of microglial. For example,at PDLcoated condition,TNF-α increased more significantly in mixed glial cells( 903. 8 ± 322. 2) ng · L~(-1) than that in microglial( 565. 4 ± 159. 8) ng·L~(-1). When cultured as a glial mixture,cells grew better on a matrigel-coated surface. However,when cultured in uncoated condition,purified microglia were more sensitive to LPS[TNF-α:( 6861. 4 ± 1606. 6) ng · L~(-1)]. Conclusions Matrigel,as a newly emerged coating material,is proved to be better than the conventional PDL-coating for culturing mixed glial cells,which improves cell viability and sensitivity. Multiplex inflammatory factor assays should be used for quantifying inflammatory response,rather than relying on q PCR alone.
Aim To revisit the long-established murine glial culture-base inflammation model,in order to explore the possible improvements of current methods. Methods The proportion and purity of glial cells were characterized by q PCR,flow cytometry and immunofluorescence. After stimulation with LPS,their inflammation response was evaluated at both mRNA and protein levels. Results Mixed glial cells were stimulated by LPS( 100μg·L~(-1)),and the expression of inflammatory factors increased more significantly than that of microglial. For example,at PDLcoated condition,TNF-α increased more significantly in mixed glial cells( 903. 8 ± 322. 2) ng · L~(-1) than that in microglial( 565. 4 ± 159. 8) ng·L~(-1). When cultured as a glial mixture,cells grew better on a matrigel-coated surface. However,when cultured in uncoated condition,purified microglia were more sensitive to LPS[TNF-α:( 6861. 4 ± 1606. 6) ng · L~(-1)]. Conclusions Matrigel,as a newly emerged coating material,is proved to be better than the conventional PDL-coating for culturing mixed glial cells,which improves cell viability and sensitivity. Multiplex inflammatory factor assays should be used for quantifying inflammatory response,rather than relying on q PCR alone.
引文
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[2]Town T,Jeng D,Alexopoulou L,et al.Microglia recognize double-stranded RNA via TLR3[J].J Immunol,2006,176(6):3804-12.
[3]Steelman A J,Li J.Poly(I:C)promotes TNF-α/TNFR1-dependent oligodendrocyte death in mixed glial cultures[J].J Neuroinflammation,2011,8:89-101.
[4]Colonna M,Butovsky O.Microglia function in the central nervous system during health and neurodegeneration[J].Annu Rev Immunol,2017,35:441-68.
[5]Roy J.Primary microglia isolation from mixed cell cultures of neonatal mouse brain tissue[J].Brain Res,2018,1689:21-9.
[6]Rodhe J.Microglia methods and protocol[M].Humana Press,2013:11-6.
[7]杨盛,何然,张飞燕,等.细胞共培养模型及其在中枢神经系统疾病研究中的应用[J].药学学报,2016,51(3):338-46.[7]Yang S,He R,Zhang F Y,et al.Application of cell co-culture techniques in central nervous system diseases[J].Acta Pharm Sin,2016,51(3):338-46.
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[9]Lin L,Desai R,Wang X Y,et al.Characteristics of primary rat microglia isolated from mixed cultures using two different methods[J].J Neuroinflammation,2017,14(1):101-10.
[10]Mizee M R,Miedema S S,van der Poel M,et al.Isolation of primary microglia from the human post-mortem brain:effects of anteand post-mortem variables[J].Acta Neuropathol Commun,2017,5(1):16-29.
[11]Timmerman R,Burm S M,Bajramovic J J.An overview of in vitro methods to study microglia[J].Front Cell Neurosci,2018,12(242):1-12.
[12]Patel A B,Tsilioni I,Leeman S E,et.al.Neurotensin stimulates sortilin and m TOR in human microglia inhibitable by methoxyluteolin,a potential therapeutic target for autism[J].Proc Natl Acad Sci USA,2016,113(45):7049-58.
[13]Haw R T,Tong C K,Yew A,et al.A three-dimensional collagen construct to model lipopolysaccharide-induced activation of BV2microglia[J].J Neuroinflammation,2014,11:134-43.
[14]Liu J,Li W,Wang Y,et al.Islet-1 overexpression in human mesenchymal stem cells promotes vascularization through monocyte chemoattractant protein-3[J].Stem Cells,2014,32(7):1843-54.
[15]Liddelow S A,Guttenplan K A,Clarke L E,et al.Neurotoxic reactive astrocytes are induced by activated microglia[J].Nature,2017,541(7638):481-7.
[16]Kettenmann H,Hanisch U K,Noda M,et al.Physiology of microglia[J].Physiol Rev,2011,91:461-553.
[17]Puyal J,Ginet V,Clarke P G.Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage:a challenge for neuroprotection[J].Prog Neurobiol,2013,105:24-48.