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本研究旨在阐明电压门控性钾通道1.3(Kv1.3)在巨噬细胞吞噬功能中的作用.利用RAW264.7巨噬细胞吞噬鸡红细胞的半定量检测系统及吞噬异硫氰酸荧光素标记的大肠杆菌(E.coli)k-12的流式细胞术定量检测系统测定巨噬细胞的吞噬功能.研究发现,用海葵神经毒素(Sh K)(100 pmol/L)选择性阻断Kv1.3通道能显著增强处于静息状态的和被脂多糖(LPS)激活的RAW264.7巨噬细胞吞噬鸡红细胞的能力;Sh K也可增强静息RAW264.7细胞吞噬大肠杆菌的能力,但由于LPS刺激吞噬的效应近乎饱和,Sh K并不能进一步增加被LPS激活的RAW264.7细胞吞噬大肠杆菌的数量.Sh K促进LPS激活的RAW264.7细胞释放一氧化氮(NO),但并不增加静息RAW264.7细胞的NO释放.Sh K(100 pmol/L)自身并不影响静息RAW264.7细胞释放细胞因子,但能抑制LPS激活的RAW264.7细胞释放白细胞介素-1?.Sh K(100 pmol/L)对RAW264.7细胞的活力无明显影响.RAW264.7细胞表达Kv1.3通道蛋白;LPS使RAW264.7细胞的Kv1.3蛋白表达下调,菲律宾菌素Ⅲ(小凹蛋白依赖性内吞途径抑制剂)使Kv1.3蛋白表达上调,细胞松弛素D对Kv1.3蛋白表达无明显影响.研究表明,RAW264.7细胞表达Kv1.3蛋白;阻断Kv1.3通道可增强RAW264.7细胞的吞噬能力和NO生成.结果提示,Kv1.3通道可能是RAW264.7细胞吞噬活动的负调节因子,有可能成为治疗巨噬细胞吞噬功能异常相关疾病的一个靶点.
This study aimed to elucidate the role of voltage-gated potassium channel1.3 (Kv1.3) in the phagocytosis of macrophages.Using RAW264.7 macrophages to phagocytose chicken erythrocytes by semi-quantitative detection system and phagocytosis of fluorescein isothiocyanate The phagocytosis of macrophages was assayed by a flow cytometry quantitative assay of labeled E. coli k-12 and found to be selectively blocked by Sh K (100 pmol / L) Kv1.3 channels significantly enhanced the ability of resting RAW264.7 macrophages to phagocytose chicken erythrocytes and LPS-activated RAW264.7 macrophages; Sh K also enhanced the ability of resting RAW264.7 cells to phagocytose E. coli, Shk did not further increase the amount of phagocytosed E. coli by LPS activated RAW264.7 cells.Sh K promoted the release of nitric oxide (NO) from RAW264.7 cells stimulated by LPS, and Did not increase NO release in resting RAW264.7 cells.ShK (100 pmol / L) did not affect resting RAW264.7 cells to release cytokines themselves, but inhibited LPS-activated RAW264.7 cells to release interleukin-1 ? Sh K (100 pmol / L) had no significant effect on the viability of RAW264.7 cells. Kv1.3 channel protein; LPS reduced Kv1.3 protein expression in RAW264.7 cells, while Filipin III (inhibitor of caveolin-dependent endocytosis pathway) upregulated Kv1.3 protein expression. Cytochalasin D up-regulated Kv1 .The expression of Kv1.3 protein in RAW264.7 cells was inhibited by blocking Kv1.3 channel.It was suggested that the Kv1.3 channel may be a part of RAW264 .7 Negative regulators of phagocytosis may be a target for the treatment of disorders associated with phagocytosis of macrophages.