目的 巨噬细胞的胞葬功能对维持机体生长发育及内环境稳定至关重要.本研究拟探讨,Nogo-B是否对巨噬细胞的胞葬功能具有调控作用及其可能机制.方法 将J774A.1小鼠巨噬细胞株分为siRNA阴性对照组与Nogo-B siRNA干扰组,采用紫外线法诱导HL60细胞凋亡.分别以绿色与红色荧光celltracker对巨噬细胞与凋亡细胞进行染色并共同孵育,免疫荧光显微镜下观察巨噬细胞对凋亡细胞的吞噬指数变化;对巨噬细胞给予凋亡细胞刺激,于刺激5、10及15 min采集巨噬细胞提取蛋白,谷胱甘肽巯基转移酶-拉下实验法检测Rac1 GTPase活性变化.结果 采用siRNA转染下调Nogo-B表达后巨噬细胞的胞葬功能明显受损,其对凋亡细胞的吞噬指数由对照组的(48.9±3.5)％显著降低至Nogo-B siRNA组的(17.8±2.1)％(P<0.01);下调Nogo-B表达后的巨噬细胞,在凋亡细胞刺激下Rac1 GTPase活性无显著变化,即出现Rac1 GTPase激活障碍,而对照组巨噬细胞在凋亡细胞刺激下Rac1 GTPase被逐渐激活.结论 Nogo-B对巨噬细胞的胞葬功能具有调控作用.下调Nogo-B表达后巨噬细胞的胞葬功能明显受损;Nogo-B可能通过Rac1 GTPase激活途径,影响细胞骨架重组,发挥对胞葬功能的调节作用.%Objective Macrophage efferocytosis plays a critical role in maintaining growth,development,and homeo-stasis.This study aimed to explore whether Nogo-B could regulate macrophage efferocytosis and the underlying mecha-nism.Methods J774A.1 mouse macrophage cell line was transfected with scramble siRNA (control)or Nogo-B siR-NA,and apoptotic HL60 cells were induced by ultraviolate radiation.Macrophage and apoptotic HL60 cells were labeled with CellTracker Green and Red respectively,then macrophages were overlaid with apoptotic cells for 2h and efferocy-tosis was analyzed using fluorescence microscopy.Proteins in Macrophage were also extracted to determine Rac1 GT-Pase activity by GST pull-down assay following stimulation with apoptotic cells.Results Down regulation of Nogo-B expression with siRNA transfection impaired macrophage efferocytosis,and the phagocytic index was reduced from (48.9±3.5)％ to (17.8±2.1)％(P<0.01).The Rac1 GTPase activity remained unchanged following apoptotic cell stimulation after down-regulation of Nogo-B expression,however its activity was gradually activated in control cells. Conclusion Nogo-B could regulate macrophage efferocytosis.Down regulation of Nogo-B expression significantly im-paired macrophage efferocytosis.Nogo-B may regulation efferocytosis through Rac1 GTPase activation pathway and therefore affecting cytoskeleton rearrangement.
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