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目的研究脂多糖诱导RAW264.7细胞产生NF-κBp65、TNF-α及诱导型一氧化氮合成酶(iNOS)的剂量、时间关系,为抗炎药物的体外筛选提供实验依据。方法调整RAW264.7细胞密度为1×109/L,种6孔板,实验设正常对照组、(1、2、5、10)μg/mL脂多糖组,免疫细胞化学方法检测NF-κBp65、iNOS及TNF-α的水平。结果 (1、2、5、10)μg/mL脂多糖可显著诱导产生NF-κBp65(P<0.05),2~10μg/mL时可显著诱导iNOS的生成(P<0.01),5~10μg/mL时可显著诱导TNF-α的生成(P<0.05)。结论 5~10μg/mL脂多糖可同时诱导产生NF-κBp65、iNOS及TNF-α,模型性能稳定,可用于多细胞因子途径的抗炎药物筛选。
Objective To study the dose and time relationship of lipopolysaccharide-induced RAW264.7 cells producing NF-κBp65, TNF-α and inducible nitric oxide synthase (iNOS), and to provide an experimental basis for in vitro screening of anti-inflammatory drugs. Methods The density of RAW264.7 cells was adjusted to 1 × 109 / L and the cells were seeded in 6-well plates. The normal control group, (1,2,5,10) μg / mL lipopolysaccharide group and the immunocytochemical method were used to detect the expression of NF-κBp65, iNOS and TNF-α levels. Results Lipopolysaccharide (1, 2, 5, 10) μg / mL could significantly induce the production of NF-κBp65 (P <0.05) mL significantly induced TNF-α production (P <0.05). Conclusion 5 ~ 10μg / mL lipopolysaccharide can induce the production of NF-κBp65, iNOS and TNF-α at the same time. The model is stable and can be used to screen anti-inflammatory drugs in multi-cytokine pathway.