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采用Western blot、免疫荧光和PCR检测小鼠单核巨噬细胞系RAW264.7中S1P受体1-3(S1PR1-3)的表达,然后应用吞噬实验和免疫荧光的方法检测磷酸鞘胺醇(sphingosine 1-phosphate,S1P)对其吞噬功能的调节。分别应用药理学工具和小干扰RNA的方法研究S1P调节其吞噬活性的作用机制。结果显示,小鼠单核巨噬细胞系RAW264.7表达S1PR1-3;S1P剂量依赖地增强小鼠单核巨噬细胞系RAW264.7的吞噬功能,应用S1PR2或S1PR3的拮抗剂和si RNAs可抑制S1P增强的小鼠单核巨噬细胞系RAW264.7的吞噬活性;而应用S1PR1的拮抗剂和si S1PR1并不影响S1P增强的RAW264.7的吞噬作用;且S1P可以显著上调RAW264.7中S1PR2和S1PR3的表达,但是不改变S1PR1的表达,提示S1P通过正反馈机制增强其介导的小鼠单核巨噬细胞系RAW264.7的吞噬功能。结果表明,S1P/S1PR2/3信号通路增强小鼠单核巨噬细胞吞噬活性,为单核巨噬细胞吞噬作用的分子机制调控研究提供了新线索。
Western blot, immunofluorescence and PCR were used to detect the expression of S1P receptor 1-3 (S1PR1-3) in mouse monocyte-macrophage cell line RAW264.7. Then phagocytosis and immunofluorescence were used to detect the expression of sphingosine phosphate sphingosine 1-phosphate, S1P) on phagocytosis. Pharmacological tools and small interfering RNA were used to study the mechanism of S1P regulating phagocytic activity. The results showed that the mouse monocyte-macrophage cell line RAW264.7 expressed S1PR1-3; S1P dose-dependently enhanced the phagocytosis of mouse monocyte-macrophage cell line RAW264.7. The antagonists of S1PR2 or S1PR3 and si RNAs Inhibition of S1P-enhanced mouse monocyte-macrophage cell line RAW264.7 phagocytic activity; and S1PR1 antagonist and si S1PR1 does not affect the S1P-enhanced phagocytosis of RAW264.7; and S1P can significantly upregulated RAW264.7 S1PR2 and S1PR3, but did not change the expression of S1PR1, suggesting that S1P enhances the phagocytosis of RAW264.7 mediated by its mouse macrophage cell line through a positive feedback mechanism. The results show that the S1P / S1PR2 / 3 signaling pathway enhances phagocytic activity of monocytes in mouse macrophages, which provides new clues for the molecular mechanism of monocyte-macrophage phagocytosis.