钙激活钾通道KCa3.1对巨噬细胞吞噬功能的抑制性调节作用

摘要

目的 观察巨噬细胞膜的钙激活钾通道KCa3.1与吞噬功能的关系.方法 通过光镜下半定量观察RAW264.7巨噬细胞吞噬鸡红细胞,以及采用流式细胞术定量检测RAW264.7巨噬细胞吞噬异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记的大肠杆菌(FITC-tagged E.coli)等方法,研究正常RAW264.7细胞的吞噬活动,以及阻断KCa3.1通道后RAW264.7细胞吞噬能力的改变.结果 光镜检测结果显示,未受吞噬刺激因子激活的对照RAW264.7细胞对鸡红细胞有较弱的吞噬能力,其吞噬率为1.67％±0.29％；用KCa3.1通道选择性阻断剂TRAM-34(20nmol/L)处理RAW264.7细胞后,吞噬率增高至5.08％±0.38％,与对照组相比差异有统计学意义(P＜0.01),且单个RAW264.7细胞吞噬鸡红细胞的个数也明显增加.流式细胞术结果显示,阻断KCa3.1通道后,RAW264.7细胞吞噬FITC标记的大肠杆菌的数量明显增多,与对照组相比差异有统计学意义(P＜0.01).结论 KCa3.1通道的活动对RAW264.7巨噬细胞的吞噬活性有抑制性调节作用.%Objective To investigate the relationship between Ca2 +-activated potassium channel KCa3.1 activity and the phagocytotic function of macrophages.Methods Microscopy and flow cytometry were used to semi-quantitatively and quantitatively estimate the phagocytotic activities of murine macrophage cell line RAW264.7 on chicken red blood cells and FITC-tagged E.coli k-12,with or without TRAM-34 (a KCa3.1 selective blocker) pre-treatment,respectively,and thus to determine the role of KCa3.1 in the phagocytosis.Results Under a light microscope,un excited RAW264.7 cells (control) showed that a weak phagocytotic activity on chicken red blood cells,the phagocytototic rate (PR) was only 1.67％ ± 0.29％.However,pre-treatment of the RAW264.7 cells with TRAM-34(20nmol/L) significantly increased the PR to 5.08％ ± 0.38％ (P ＜ 0.01 vs control).In the flow cytometry assay,blocking KCa3.1 with TRAM-34 also significantly increased the phagocytotic activities of RAW264.7 cells on FITC-tagged E.coli k-12 compared with the control (P ＜ 0.01).Conclusion The activity of KCa3.1 exerts an inhibitory role on the phagocytotic function of RAW264.7 macrophages.