流式微球分析法检测小檗碱对RAW264.7细胞分泌炎症因子的影响

Detection of Inflammatory Cytokines Secreted by LPS-Stimulated RAW264.7 Cells by Means of Cytometric Bead Array

建立并优化了在FACSAriaⅠ流式细胞仪上应用流式微球分析法(CBA法)检测分泌型炎症细胞因子的方法。用CBA法检测小檗碱对经LPS诱导的小鼠RAW264.7细胞分泌IL-6I、L-10I、FN-γ、MCP-1、TNFI、L-12p70等细胞因子的影响。结果显示,小檗碱可显著抑制LPS诱导的各种炎症细胞因子的分泌,并呈良好的量效关系。12 mol/L的小檗碱对IL-10I、FN-、TNF及IL-12 p70的抑制率分别为5.10±0.24%、18.64±3.95%、22.59±0.56%、68.27±2.49%、48 mol/L的小檗碱抑制作用更显著,抑制率分别为45.62±1.51%、39.00±3.58%、79.61±5.17%9、2.03±0.85%。各细胞因子荧光值与其实际浓度具良好的线性关系,相关系数均在0.999以上。流式微球分析法是一种灵敏高效、节省成本、重复性好的检测方法,为肿瘤及免疫相关炎症因子的高通量检测提供了现实可行的方法。

Cytometric bead array（CBA） method for the detection of secreted cytokines was established and optimized on FACSAria I cytometer. The concentrations of IL-6,IL-10,MCP-1 ,TNF,IFN-γ,IL12p70 cytokines secreted by LPS-stimulated R.AW264.7 cells were detected by means of CBA. Berberine can significantly down-regulate secretion of various inflammatory cytokines. The inhibitory rate of 12μmol/L berberine against IL-10, IFN-γ, TNF, IL-12 p70 were 5.10±0.24% ,18.64±3.95%, 22.59±0.56%, 68.27±2.49%, respectively. 13erberine at 48μmol/L remarkably inhibited the increasing expression of IL-10, IFN-y, TNF, IL-12 p70 stimulated by LPS by 45.62±1.51%, 39.00± 3.58%, 79.61±5.17%, 92.03±0.85%. CBA assay is a realistic high-throughput approach for the detection of inflammatory cytokines because of its sensitivity, efficiency, cost-effectiveness and reproducibility.